Fusion molecules and methods for treatment of immune diseases

ABSTRACT

The invention concerns bifunctional fusion molecules, and novel, safer and more efficacious methods for the treatment of immune disorders resulting from excessive or unwanted immune responses. The invention provides methods for the suppression of type I hypersensitive (i.e., IgE-mediated) allergic conditions, methods for the prevention of anaphylactic responses that occur as a result of traditional peptide immunotherapies for allergic and autoimmune disorders, and provides novel methods for the treatment of autoimmune conditions, where the methods have reduced risk of triggering an anaphylactic response. The invention provides novel therapeutic approaches for the treatment of allergic responses, including the prevention of anaphylactic response that can occur from environmental allergen exposure. The invention also provides methods for the treatment of autoimmune disorders such as multiple sclerosis, autoimmune type I diabetes mellitus, and rheumatoid arthritis. The invention also provides methods for preventing anaphylactic response during traditional antigen therapies.

[0001] This application is a continuation-in-part application claimingpriority under 35 U.S.C. §120 to copending U.S. patent application Ser.No. 09/847,208, filed May 1, 2001, which is hereby incorporated byreference in its entirety.

[0002] This invention was made with Government support under Grant No.A115251, awarded by the National Institutes of Health. The Governmenthas certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The invention concerns a new approach for the management ofimmune diseases using novel fusion polypeptides. More specifically, theinvention is related to the treatment of immune diseases, wheremanagement of the disease comprises suppressing an inappropriate orunwanted immune response, such as, for example, autoimmune diseases andallergic diseases.

[0005] 2. Description of the Related Art

[0006] Immunoglobulin Receptors

[0007] Immunoglobulin receptors (also referred to as Fc receptors) arecell-surface receptors binding the constant region of immunoglobulins,and mediate various immunoglobulin functions other than antigen binding.

[0008] Fc receptors for IgE molecules are found on many cell types ofthe immune system (Fridman, W., FASEB J., 5(12):2684-90 (1991)). Thereare two different receptors currently known for IgE. IgE mediates itsbiological responses as an antibody through the multichain high-affinityreceptor, FcεRI, and the low-affinity receptor, FcεRII. Thehigh-affinity FcεRI, expressed on the surface of mast cells, basophils,and Langerhans cells, belongs to the immunoglobulin gene superfamily,and has a tetrameric structure composed of an α-chain, a β-chain and twodisulfide-linked γ-chains (Adamczewski, M., and Kinet, J. P., ChemicalImmun., 59:173-190 (1994)) that are required for receptor expression andsignal transduction (Tunon de Lara, Rev. Mal. Respir., 13(1):27-36(1996)). The α-chain of the receptor interacts with the distal portionof the third constant domain of the IgE heavy chain. The specific aminoacids of human IgE involved in binding to human FcεR1 have beenidentified as including Arg-408, Ser-41 1, Lys-415, Glu-452, Arg-465,and Met-469 (Presta et al., J. Biol. Chem. 269:26368-73 (1994)). Theinteraction is highly specific with a binding constant of about 10¹⁰M⁻¹.

[0009] The low-affinity FcεRII receptor, represented on the surface ofinflammatory cells, including eosinophils, leukocytes, B lymphocytes,and platelets, did not evolve from the immunoglobulin superfamily buthas substantial homology with several animal lectins (Yodoi et al., CibaFound. Symp., 147:133-148 (1989)) and is made up of a transmembranechain with an intracytoplasmic NH₂ terminus. The low-affinity receptor,FcεRI (CD23) is currently known to have two forms (FcRIIa and FcεRIIb),both of which have been cloned and sequenced. They differ only in theN-terminal cytoplasmic region, the extracellular domains beingidentical. FcεRIIa is normally expressed on B cells, while FcεRIIb isexpressed on T cells, B cells, monocytes and eosinophils upon inductionby the cytokine IL-4.

[0010] Through the high-affinity IgE receptor, FcεRII, IgE plays keyroles in an array of acute and chronic allergic reactions, includingasthma, allergic rhinitis, atopic dermatitis, severe food allergies,chronic urticaria and angioedema, as well as the serious physiologicalcondition of anaphylactic shock as results, for example, from bee stingsor penicillin allergy. Binding of a multivalent antigen (allergen) toantigen-specific IgE specifically bound to FcεRI on the surface of mastcells and basophils stimulates a complex series of signaling events thatculminate in the release of host vasoactive and proinflammatorymediators contributing to both acute and late-phase allergic responses(Metcalfe et al., Physiol. Rev. 77:1033-1079 (1997)).

[0011] The function of the low affinity IgE receptor, FcεRII (alsoreferred to as CD23), found on the surface of B lymphocytes, is muchless well established than that of FcεRI. FcεRII, in a polymeric state,binds IgE, and this binding may play a role in controlling the type(class) of antibody produced by B cells.

[0012] Three groups of receptors that bind the constant region of humanIgG have so far been identified on cell surfaces: FcγRI (CD64), FcγRII(CD32), and FcγRIII (CD16), all of which belong to the immunoglobulingene superfamily. The three Fcγ receptors have a large number of variousisoforms. Along with the stimulatory FcεRI, mast cells and basophilsco-express an immunoreceptor tyrosine-based inhibition motif(ITIM)-containing inhibitory low-affinity receptor, FcγRIIb, that actsas a negative regulator of antibody function. FcγRIIb represents agrowing family of structurally and functionally similar inhibitoryreceptors, the inhibitory receptor superfamily (IRS), that negativelyregulate immunoreceptor tyrosine-based activation motif(ITAM)-containing immune receptors (Ott and Cambier, J. Allergy Clin.Immunol., 106:429-440 (2000)) and a diverse array of cellular responses.Coaggregation of an IRS member with an activating receptor leads tophosphorylation of the characteristic ITIM tyrosine and subsequentrecruitment of the SH2 domain-containing protein tyrosine phosphatases,SHP-1 and SHP-2, and the SH2 domain-containing phospholipases, SHIP andSHIP2 (Cambier, J. C., Proc. Natl. Acad. Sci. USA, 94:5993-5995 (1997)).Possible outcomes of the coaggregation include inhibition of cellularactivation, as demonstrated by the coaggregation of FcγRIIb and B-cellreceptors, T-cell receptors, activating receptors, including FcεR1, orcytokine receptors (Malbec et al., Curr. Top. Microbiol. Immunol.,244:13-27 (1999)).

[0013] Most studies have so far concentrated on elucidating themechanisms of FcγRII, in particular, FcγRIIb function. The threealternatively spliced isoforms of the FcγIIb receptor, of which FcγRIIb1is only found in mice, and FcγRIIb1 and FcγRIIb2 are expressed in bothhumans and mice, have Ig-like loops and a conserved ITIM, but differ intheir cytoplasmic domains. Co-crosslinking of the high-affinity FcεR1receptor and the inhibitory low-affinity receptor FcγRII blocks a numberof processes, including FcεRI-mediated secretion, IL-4 production, Ca²⁺mobilization, Syk phosphorylation, and FcεRI-mediated basophil and mastcell activation. In B cells, co-crosslinking of the B-cell receptor andFcγRIIb inhibits B-cell receptor-mediated cell activation (Cambier, J.C., Proc. Natl. Acad. Sci., 94:5993-5995 (1997); Daeron, M., Annu. Rev.Immunol, 15:203-234 (1997)), and specifically, inhibits B-cellreceptor-induced blastogenesis and proliferation (Chan et al.,Immunology, 21:967-981 (1971); Phillips and Parker, J. Immunol.,132:627-632 (1984)) and stimulates apoptosis (Ashman et al., J. Immunol,157:5-1 1 (1996)). Coaggregation of FcγRIIb1 or FcγRIIb2 with FcεR1 inrat basophilic leukemia cells, inhibits FcεRI-mediated release ofserotonin and TNF-α (Daeron et al., J. Clin. Invest., 95:577-85 (1995);Daeron et al., Immunity, 3:635-646 (1995)).

[0014] Another ITIM-containing receptor expressed on mast cells that hasbeen described to prevent IgE-mediated mast cell activation whencoligated with FcεRI, is a 49 kDa glycoprotein member of theimmunoglobulin superfamily, termed gp49b1 (gp91) (see, e.g., Wagtmann etal., Current Top. Micobiol. Immunol. 244:107-113 (1999); Katz, H. R.,Int. Arch Allergy Immunol. 118:177-179 (1999); and Lu-Kuo et al., J.Biol. Chem. 274:5791-96 (1999)). Gp49b1 was originally identified inmice, while human counterparts of the gp49 family, including gp49b1,have been cloned by Arm et al., J. Immunol. 15:2342-2349 (1997). FurtherITIM-containing receptors, several expressed in mast cells, basophils orB cells are reviewed by Sinclair N R, Scand. J. Immunol., 50:10-13(1999).

[0015] Through the high-affinity IgE receptor FcεRI, IgE plays key rolesin immune response. The activation of mast cells and basophils byantigen (i.e., allergen) via an antigen-specific IgE/FcεRI pathwayresults in the release of host vasoactive and proinflammatory mediators(i.e., degranulation), which contributes to the allergic response(Oliver et al., Immunopharmacology 48:269-281 [2000]; Metcalfe et al.,Physiol. Rev., 77:1033-1079 [1997]). These and other biochemical eventslead to the rapid secretion of inflammatory mediators such as histamine,resulting in physiological responses that include localized tissueinflammation, vasodilation, increased blood vessel and mucosalpermeability, and local recruitment of other immune system cells,including additional basophils and mast cells. In moderation, theseresponses have a beneficial role in immunity against parasites and othermicroorganisms. However, when in excess, this physiological responseresults in the varied pathological conditions of allergy, also known astype I hypersensitivity.

[0016] Allergic Conditions

[0017] Allergy is manifested in a broad array of conditions andassociated symptoms, which may be mild, chronic, acute and/or lifethreatening. These various pathologies include, for example, allergicasthma, allergic rhinitis, atopic dermatitis, severe food allergies,chronic urticaria and angioedema, as well as the serious physiologicalcondition of anaphylactic shock. A wide variety of antigens are known toact as allergens, and exposure to these allergens results in theallergic pathology. Common allergens include, but are not limited to,bee stings, penicillin, various food allergies, pollens, animal detritus(especially house dust mite, cat, dog and cockroach), and fungalallergens. The most severe responses to allergens can result in airwayconstriction and anaphylactic shock, both of which are potentially fatalconditions. Despite advances in understanding the cellular and molecularmechanisms that control allergic responses and improved therapies, theincidence of allergic diseases, especially allergic asthma, hasincreased dramatically in recent years in both developed and developingcountries (Beasley et al., J. Allergy Clin. Immunol. 105:466-472 (2000);Peat and Li, J. Allergy Clin. Immunol. 103:1-10 (1999)). Thus, thereexists a strong need to develop treatments for allergic diseases.

[0018] Allergic asthma is a condition brought about by exposure toubiquitous, environmental allergens, resulting in an inflammatoryresponse and constriction of the upper airway in hypersensitiveindividuals. Mild asthma can usually be controlled in most patients byrelatively low doses of inhaled corticosteroids, while moderate asthmais usually managed by the additional administration of inhaledlong-acting P-antagonists or leukotriene inhibitors. The treatment ofsevere asthma is still a serious medical problem. In addition, many ofthe therapeutics currently used in allergy treatment have seriousside-effects. Although an anti-IgE antibody currently in clinical trials(rhuMAb-E25, Genentech, Inc.) and other experimental therapies (e.g.,antagonists of IL-4) show promising results, there is need for thedevelopment of additional therapeutic strategies and agents to controlallergic disease, such as asthma, severe food allergy, and chronicurticaria and angioedema.

[0019] One approach to the treatment of allergic diseases is by use ofallergen-based immunotherapy. This methodology uses whole antigens as“allergy vaccines” and is now appreciated to induce a state of relativeallergic tolerance. This technique for the treatment of allergy isfrequently termed “desensitization” or “hyposensitization” therapy. Inthis technique, increasing doses of allergen are administered, typicallyby injection, to a subject over an extended period of time, frequentlymonths or years. The mechanism of action of this therapy is thought toinvolve induction of IgG inhibitory antibodies, suppression of mastcell/basophil reactivity, suppression of T-cell responses, the promotionof T-cell anergy, and/or clonal deletion, and in the long term, decreasein the levels of allergen specific IgE. The use of this approach is,however, hindered in many instances by poor efficacy and seriousside-effects, including the risk of triggering a systemic andpotentially fatal anaphylactic response, where the clinicaladministration of the allergen induces the severe allergic response itseeks to suppress (TePas et al., Curr. Opin. Pediatrics 12:574-578[2000]).

[0020] Refinements of this technique use smaller portions of theallergen molecule, where the small portions (i.e., peptides) presumablycontain the immunodominant epitope(s) for T cells regulating theallergic reaction. Immunotolerance therapy using these allergenicportions is also termed peptide therapy, in which increasing doses ofallergenic peptide are administered, typically by injection, to asubject. The mechanism of action of this therapy is thought to involvesuppression of T-cell responses, the promotion of T-cell anergy, and/orclonal deletion. Since the peptides are designed to bind only to T cellsand not to allergic (IgE) antibodies, it was hoped that the use of thisapproach would not induce allergic reactions to the treatment.Unfortunately, these peptide therapy trials have met withdisappointment, and allergic reactions are often observed in response tothe treatments. Development of these peptide therapy methods havelargely been discontinued.

[0021] Autoimmune Diseases

[0022] It is estimated that as much as 20 percent of the Americanpopulation has some type of autoimmune disease. Autoimmune diseasesdemonstrate disproportionate expression in women, where it is estimatedthat as many as 75% of those affected with autoimmune disorders arewomen. Although some forms of autoimmune diseases are individually rare,some diseases, such as rheumatoid arthritis and autoimmune thyroiditis,account for significant morbidity in the population (Rose and MacKay(Eds.), The Autoimmune Diseases, Third Edition, Academic Press [1998]).

[0023] Autoimmune disease results from failure of the body to eliminateself-reactive T-cells and B-cells from the immune repertoire, resultingin circulating B-cell products (i.e., autoreactive antibodies) andT-cells that are capable of identifying and inducing an immune responseto molecules native to the subject's own physiology. Particularautoimmune disorders can be generally classified as organ-specific(i.e., cell-type specific) or systemic (i.e., non-organ specific), butwith some diseases showing aspects of both ends of this continuum.Organ-specific disorders include, for example, Hashimoto's thyroiditis(thyroid gland) and insulin dependent diabetes mellitus (pancreas).Examples of systemic disorders include rheumatoid arthritis and systemiclupus erythematosus. Since an autoimmune response can potentially begenerated against any organ or tissue in the body, the autoimmunediseases display a legion of signs and symptoms. Furthermore, when bloodvessels are a target of the autoimmune attack as in the autoimmunevasculitides, all organs may be involved. Autoimmune diseases display awide variety of severity varying from mild to life-threatening, and fromacute to chronic, and relapsing (Rose and MacKay (Eds.), The AutoimmuneDiseases, Third Edition, Academic Press [1998]; and Davidson andDiamond, N. Engl. J. Med., 345(5):340-350 [2001]).

[0024] The molecular identity of some of the self-reactive antigens(i.e., the autoantigen) are known in some, but not all, autoimmunediseases. The diagnosis and study of autoimmune diseases is complicatedby the promiscuous nature of these disorders, where a patient with anautoimmune disease can have multiple types of autoreactive antibodies,and vice versa, a single type of autoreactive antibody is sometimesobserved in multiple autoimmune disease states (Mocci et al., Curr.Opin. Immunol., 12:725-730 [2000]; and Davidson and Diamond, N. Engl. J.Med., 345(5):340-350 [2001]). Furthermore, autoreactive antibodies orT-cells may be present in an individual, but that individual will notshow any indication of disease or other pathology. Thus while themolecular identity of many autoantigens is known, the exact pathogenicrole of these autoantigens generally remains obscure (with notableexceptions, for example, myesthenia gravis, autoimmune thyroid disease,multiple sclerosis and diabetes mellitus).

[0025] Treatments for autoimmune diseases exist, but each method has itsown particular drawbacks. Existing treatments for autoimmune disorderscan be generally placed in two groups. First, and of most immediateimportance, are treatments to compensate for a physiological deficiency,typically by the replacement of a hormone or other product that isabsent in the patient. For example, autoimmune diabetes mellitus can betreated by the administration of insulin, while autoimmune thyroiddisease is treated by giving thyroid hormone. Treatments of otherdisorders entails the replacement of various blood components, such asplatelets in immune thrombocytopenia or use of drugs (e.g.,erythropoetin) to stimulate the production of red blood cells in immunebased anemia. In some cases, tissue grafts or mechanical substitutesoffer possible treatment options, such as in lupus nephritis and chronicrheumatoid arthritis. Unfortunately, these types of treatments aresuboptimal, as they merely alleviate the disease symptoms, and do notcorrect the underlying autoimmune pathology and the development ofvarious disease related complications. Since the underlying autoimmuneactivity is still present, affected tissues, tissue grafts, orreplacement proteins are likely to succumb to the same immunedegeneration.

[0026] The second category of autoimmune disease treatments are thosetherapies that result in generalized suppression of the inflammatory andimmune response. This approach is difficult at best, as it necessitatesa balance between suppressing the disease-causing immune reaction, yetpreserving the body's ability to fight infection. The drugs mostcommonly used in conventional anti-inflammatory therapy to treatautoimmune disorders are the non-steroidal anti-inflammatory drugs(e.g., aspirin, ibuprofen, etc). Unfortunately, these drugs simplyrelieve the inflammation and associated pain and other symptoms, but donot modify progression of the disease. Broad acting immunosuppressants,such as cyclosporine A, azathioprine, cyclophosphamide, andmethotrexate, are commonly used to treat symptoms as well as hopefullyameliorate the course of the autoimmune process. Although somewhatsuccessful in controlling the autoimmune tissue injury, these broadacting and powerful drugs often have severe side effects, such as thedevelopment of neoplasias, destruction of bone marrow and other rapidlydividing cells and tissues, and risk of liver and kidney injury.Furthermore, these drugs have the undesirable consequence of depressingthe patient's immune system, which carries the risk of severe infectiouscomplications. For these reasons, general suppression of the immunesystem is generally reserved for the treatment of severe autoimmunedisorders, such as dermatomyositis and systemic lupus erythematosus(SLE) or when there is involvement of a critical organ, such as theheart.

[0027] More preferably, successful immuno-suppressive therapies forautoimmune disorders will suppress the immune system in anautoantigen-specific manner (i.e., antigen-restricted tolerance),similar to that proposed for allergen immunotolerance therapy to inducedesensitization (Harrison and Hafler, Curr. Opin. Immunol., 12:704-711[2000]; Weiner, Annu. Rev. Med., 48:341-351 [1997]; and Mocci et al.,Curr. Opin. Immunol., 12:725-730 [2000]). Refinements of this approachhave used smaller portions of the autoantigen (i.e., autoantigenicpeptides) which contain the immunodominant epitope(s), using oral andparenteral administration protocols. Like allergy peptide therapies,administration of autoantigen peptides is now recognized to beaccompanied by significant risk of allergic/hypersensitivity reactionsand potentially fatal anaphylactic response. These risks also limit theamount of peptide that can be administered in a single dose. For theseand other reasons, peptide immunotolerance therapies for the treatmentof autoimmune diseases in humans have been problematic, and many havefailed to find widespread applicability. These tolerance therapiesremain largely unusable, unless the risk of allergic reactions can beovercome.

[0028] Autoimmune type-I diabetes mellitus is a form ofinsulin-dependent diabetes resulting from immune recognition of insulinor those cells that produce insulin, i.e., the pancreatic islet β-cells,leading to immune-mediated destruction of the β-cells, and reduction ofinsulin production or activity. The disease is thought to be initiatedby multiple etiologies, but all resulting in insulin deficiency. Theknown autoantigen targets of autoimmune diabetes include insulin andglutamic acid decarboxylase (GAD) (Chaillous et al., Diabetologia37(5):491-499 [1994]; Naquet et al., J. Immunol., 140(8):2569-2578[1988]; Yoon et al., Science 284(5417):1183-1187 [1999]; Nepom et al.,Proc. Natl. Acad. Sci. USA 98(4):1763-1768 [2001]). In addition toinsulin and GAD, additional β-cell autoantigens are theorized to exist(Nepom, Curr. Opin. Immunol., 7(6):825-830 [1995]).

[0029] Tolerance therapies incorporating either parenterally and orallyadministered diabetes autoantigens (including insulin and GAD) have beentried in experimental models and human subjects. However, the majorityof human trials have met with disappointment. Furthermore, widespreadapplication of peptide therapy in humans to treat autoimmune diabeteshas been prevented by the observation that in some cases, peptideadministration may actually accelerate disease progression (Pozzilli etal., Diabetologia 43:1000-1004 [2000]; Gale, Lancet 356(9229):526-527[2000]; Chaillous et al., Lancet 356:545-549 [2000]; Blanas et al.,Science 274:1707-1709 [1996]; McFarland, Science 274(5295):2037 [1996];and Bellmann et al., Diabetologia 41:844-887 [1998]).

[0030] Rheumatoid arthritis (RA) is another severe autoimmune disorderthat impacts a significant percentage of the population. RA is asystemic disease characterized by chronic inflammation primarily of thesynovial membrane lining of the joints, although the disease can effecta host of other tissues, such as the lung. This joint inflammation leadsto chronic pain, loss of function, and ultimately to destruction of thejoint. The presence of T-cells in the synovia, as well as other lines ofevidence, indicate an autoimmune disease etiology. A number ofautoantigen candidates for this disease have been tentativelyidentified, including type II collagen, human cartilage protein gp39 andgp130-RAPS. Existing treatment regimens for RA include anti-inflammatorydrugs (both steroidal and non-steroidal), cytotoxic therapy (e.g.,cyclosporine A, methotrexate and leflunomide), and biological immunemodulators such as interleukins-1 and -2 receptor antagonists,anti-tumor necrosis factor alpha (TNFα) monoclonal antibodies, and TNFαreceptor-IgG1 fusion proteins, frequently in conjunction withmethotrexate (Davidson and Diamond, N. Engl. J. Med., 345(5):340-350[2001]). However, these biological modifier therapies are suboptimal fora variety of reasons, notably do to their limited effectiveness andtoxicity such as the systemic cytokine release syndrome seen withadministration of a number of cytokines (e.g., IL-2), or the recentlyrecognized increased risk of infection with anti-TNFα treatments.

[0031] In T-cells isolated from patients with this disease, it has beenobserved that some T-cell receptor (TCR) β-subunit variable domains (Vi)appear to be preferentially utilized compared to disease-free subjects.It is suggested that peptides corresponding to these preferentiallyutilized TCR V_(β) domains can be used in peptide vaccination therapy,where vaccination will result in disease-specific anti-TCR antibodies,and hopefully alleviate the disease (Bridges and Moreland, Rheum. Dis.Clin. North Am., 24(3):641-650 [1998]; and Gold et al., Crit. Rev.Immunol., 17(5-6):507-510 [1997]). This therapy is under development(Moreland et al., J. Rheumatol., 23(8):1353-1362 [1996]; and Moreland etal., Arthritis Rheum., 41(11):1919-1929 [1998]), but has proven to beproblematic due to the lack of consistency in TCR use in humans asopposed to what was observed in experimental animals.

[0032] A proposed alternative to antibody-based therapies for rheumatoidarthritis and other autoimmune diseases are therapies that incorporatemajor histocompatibility complex class II proteins (MHC II) covalentlycoupled with autoreactive peptides (Sharma et al., Proc. Natl. Acad.Sci. USA 88:11465-11469 [1991]; and Spack et al., Autoimmunity 8:787-807[1995]). A variation of this MHC-based therapy incorporates covalentlycoupled Fcγ domains for the purpose of producing dimeric MHC/antigenfusion polypeptides (Casares et al., Protein Eng., 10(11):1295-1301[1997]; and Casares et al., J. Exp. Med., 190(4):543-553 [1999]).However, these approaches based on artificial antigen presentation inthe context of an MHC II fusion protein are unlikely to be widelyapplicable in human systems, as the MHC loci in humans are multiallelic(i.e., there exist many haplotype variations).

[0033] Another autoimmune disorder impacting a significant portion ofthe population is multiple sclerosis (MS), which afflicts approximately250,000 people in the United States alone. MS manifests mainly inadults, and displays a wide array of neurological-related symptoms thatvary unpredictably over decades, and may relapse, progress, or undergospontaneous remission. No therapies currently exist that can arrest theprogression of the primary neurologic disability caused by MS. Currenttherapies favor the use of glucocorticosteroids, but unfortunatelycorticosteroid therapies are not believed to alter the long-term courseof the disease. Furthermore, corticosteroids have many side effects,including increased risk of infection, osteoporosis, gastric bleeding,cataracts and hypertension. Immunosuppressants are sometimes tried inprogressive MS, but with equivocal results. Biological immunemodulators, such as interferons α and β1a, and copolymer I, have alsobeen tried in an attempt to downregulate the immune response and controlthe progression of the disease. Administration of interferon-β tosuppress general immune function in patients with multiple sclerosis hashad some limited success (Rose and MacKay (Eds.), The AutoimmuneDiseases, Third Edition, Academic Press, p.572-578 [1998]; Davidson andDiamond, N. Engl. J. Med., 345(5):340-350 [2001]). However, thesebiological modifiers have the drawback of limited efficacy and systemicside effects of fever and flu-like reactions.

[0034] The varied neurological-related symptoms of MS are the result ofdegeneration of the myelin sheath surrounding neurons within the centralnervous system (CNS), as well as loss of cells that deposit and supportthe myelin sheaths, i.e., the oligodendrocytes, with ensuing damage tothe underlying axons. T-cells isolated from patients with MS respond tomyelin-basic-protein (MBP) by proliferating and secretingproinflammatory cytokines, indicating that endogenous MBP is at leastone of the autoantigens being recognized in patients with the disease.The immunodominant epitope on the MBP protein has been shown to residewithin the MBP₈₃₋₉₉ region. As is the case in many autoimmune diseases,at least one other autoantibody has been implicated as the causativeagent in patients with multiple sclerosis. This autoantibody appears tobe specific for myelin oligodendrocyte glycoprotein (MOG), with adominant epitope at MOG₉₂₋₁₀₆.

[0035] Peptide immunotherapies using the MBP epitope to treat MS havebeen tested in animal models and in humans (e.g., Weiner et al., Science259(5099):1321-1324 [1993]; Warren et al., Jour. Neuro. Sci., 152:31-38[1997]; Goodkin et al., Neurology 54:1414-1420 [2000]; Kappos et al.,Nat. Med., 6(10):1176-1182 [2000]; Bielekova et al., Nat. Med.,6(10):1167-1175 [2000]; and Steinman and Conlon, Jour. Clin. Immunol.,21(2):93-98 [2001]). Unfortunately, those studies using human subjectshave been disappointing, with significant toxicity and hypersensitivityreactions reported. Furthermore, multiple sclerosis autoantigenimmunotherapy may actually exacerbate the disease in some cases(McFarland, Science 274(5295):2037 [1996]; and Genain et al., Science274:2054-2057 [1996]).

[0036] What is needed are improved and/or novel therapeutic strategiesfor the treatment of immune diseases resulting from inappropriate orunwanted immune response. What are needed are methods for the treatmentof autoimmune diseases that are widely applicable to many autoimmunediseases, do not have the toxic effects of broad immunosuppressantdrugs, and act in an autoantigen-restricted manner, thereby preserving apatent's immune function. Accordingly, there is a need for improvedmethods for peptide tolerance immunotherapies that have reduced risk ofhypersensitivity reactions, and most notably, anaphylactic responses.Similarly, there is a need for compositions and methods that permithigher dosages of traditional peptide tolerance therapies, without therisk of inducing hypersensitivity responses.

[0037] The object of this invention is to provide novel and/or improvedtherapeutic strategies for the treatment of immune diseases resultingfrom inappropriate or unwanted immune response. Allergic diseases andautoimmune diseases are two such types of diseases which can be treatedwith the compositions and methods provided by the present invention.Allergic diseases which may be treated using the invention include, butare not limited to, for example, atopic allergies such as asthma,allergic rhinitis, atopic dermatitis, severe food allergies, some formsof chronic urticaria and angioedema, as well as the seriousphysiological condition of anaphylactic shock (i.e., anaphylactichypersensitivity) resulting from, for example, bee stings or penicillinallergy. Autoimmune diseases which can be treated using the presentinvention include, but are not limited to, autoimmune diabetes,rheumatoid arthritis, and multiple sclerosis, for example.

[0038] The methods for treating allergic and autoimmune diseasesprovided by the invention can also be used in conjunction withtraditional peptide immunotherapies, where the fusion moleculesdescribed herein are administered before, during or after the peptideimmunotherapy, and find particular use in preventing the anaphylacticreactions associated with traditional immunotherapies.

SUMMARY OF THE INVENTION

[0039] The present invention provides novel multi-functional compoundsthat have the ability to crosslink inhibitory receptors with Fcεreceptors and block Fcε receptor-mediated biological activities, as wellas methods for using such compounds, and compositions and articles ofmanufacture comprising them. The invention also provides compositionsand methods suitable for the prevention or treatment of immune-mediateddiseases.

[0040] One aspect the invention concerns an isolated fusion moleculecomprising a first polypeptide sequence capable of specific binding, toa native inhibitory receptor comprising an immune receptortyrosine-based inhibitory motif (ITIM), functionally connected to asecond polypeptide sequence capable of specific binding, through a thirdpolypeptide sequence, to a native IgE receptor (FcεR), wherein the firstand second polypeptide sequences are other than antibody variableregions, and wherein said fusion molecule is not capable of T cellinteraction prior to internalization. Preferably, the second polypeptidesequence comprises an antigen sequence, and more preferably, at least aportion of an autoantigen sequence. In one embodiment, the autoantigensequence comprises at least one autoantigenic epitope. In one preferredembodiment, the third polypeptide is an immunoglobulin specific for theautoantigen. In a particularly preferred embodiment, the immunoglobulinspecific for the autoantigen is an IgE class antibody.

[0041] In some preferred embodiments, the autoantigen sequence in thefusion molecule is selected from the group consisting of rheumatoidarthritis autoantigen, multiple sclerosis autoantigen, or autoimmunetype I diabetes mellitus autoantigen, and portions thereof. In otherpreferred embodiments, the autoantigen is selected from the groupconsisting of myelin basic protein (MBP), proteolipid protein, myelinoligodendrocyte glycoprotein, β-crystallin, myelin-associatedglycoprotein, Po glycoprotein, PMP22, 2′,3′-cyclic nucleotide3′-phosphohydrolase (CNPase), glutamic acid decarboxylase (GAD),insulin, 64 kD islet cell antigen (IA-2, also termed ICA512), phogrin(IA-2β), type II collagen, human cartilage gp39 (HCgp39), andgp130-RAPS, and portions thereof.

[0042] In other preferred embodiments, the autoantigen sequence in thefusion molecule comprises at least 90% sequence identity with at least aportion of an autoantigen sequence. In still other preferredembodiments, the autoantigen sequence in the fusion molecule comprisesan amino acid sequence encoded by a nucleic acid hybridizing understringent conditions to at least a portion of the complement of anucleic acid molecule encoding an autoantigen.

[0043] In a particularly preferred embodiments, the inhibitory receptoris a type I transmembrane molecule with an Ig-like domain, such as, forexample, a low-affinity FcγRIIb IgG receptor, and the IgE receptor maybe a FcεRI high-affinity receptor or a low-affinity FCεRII receptor(CD23). More preferably, the FcγRIIb and FcεRI receptors are of humanorigin. In a related embodiment, the first polypeptide sequencecomprises an amino acid sequence having at least 85% identity with anative human IgG heavy chain constant region sequence. Indeed, the IgGportion of the molecule can derive from the heavy chain constant regionof any IgG subclass, including IgG₁, IgG₂, IgG₃ and IgG₄. Furthermore,the native human IgG heavy chain constant region sequence can be thenative human IgG heavy chain constant region sequence of SEQ ID NO: 2.

[0044] In another embodiment, the first polypeptide sequence comprisespreferably an amino acid sequence having at least 85% identity to thehinge-CH2-CH3 domain amino acid sequence of SEQ ID NO: 3, and morepreferably, at least 90% identity, and more preferably still, at least95% identity, and most preferably, at least 98% identity. In still otherembodiments, the first polypeptide comprises a least part of the CH2 andCH3 domains of a native human IgG₁ constant region, or additionallycomprises a least part of the hinge of a native human IgG₁ constantregion. Alternatively, the first polypeptide sequence comprises at leastpart of the hinge, CH2 and CH3 domains of a native human IgG₁ heavychain constant region in the absence of a functional CH1 region, andalternatively still, the first polypeptide sequence comprises an aminoacid sequence encoded by a nucleic acid hybridizing under stringentconditions to at least a portion of the complement of the IgG heavychain constant region nucleotide sequence of SEQ ID NO: 1.

[0045] In some embodiments, the first and second polypeptide sequencesmay be functionally connected via a linker, e.g., a polypeptide linker.The length of the polypeptide linker typically is about 5 to 25 aminoacid residues. In one embodiment, the polypeptide linker comprises atleast one proteasome proteolysis signal, wherein the signal is selectedfrom the group consisting of large hydrophobic amino acid residues,basic amino acid residues and acidic amino acid residues. In otherembodiments, the polypeptide linker sequence comprises at least oneendopeptidase recognition motif. In other embodiments, the polypeptidelinker sequence comprises a plurality of endopeptidase recognitionmotifs, and these endopeptidase motifs may include cysteine, aspartateor asparagine amino acid residues. In other embodiments, the fusionmolecule comprises at least one amino-terminal ubiquitination targetmotif. In still other embodiments, the fusion molecule can display atleast one proteasome proteolysis signal, wherein that signal is selectedfrom the group consisting of large hydrophobic amino acid residues,basic amino acid residues or acidic amino acid residues.

[0046] In a further aspect, the present invention provides isolatednucleic acid molecules encoding a fusion molecule comprising a firstpolypeptide sequence capable of specific binding, to a native inhibitoryreceptor comprising an immune receptor tyrosine-based inhibitory motif(ITIM), functionally connected to a second polypeptide sequence that isan autoantigen sequence capable of specific binding, through a thirdpolypeptide sequence, to a native IgE receptor (FcεR), wherein the firstand second polypeptide sequences are other than antibody variableregions, and wherein said fusion molecule is not capable of T cellinteraction prior to internalization. The invention also providesvectors and host cells comprising these nucleic acids. Similarly, thepresent invention provides isolated nucleic acid molecules as describedabove, wherein the second polypeptide sequence in the fusion moleculeencodes at least a portion of an autoantigen. Vectors and host cellscomprising these nucleic acids are also encompassed by the presentinvention.

[0047] In a further aspect, the invention concerns a pharmaceuticalcomposition comprising a fusion molecule as hereinabove defined inadmixture with a pharmaceutically acceptable excipient or ingredient. Ina still further aspect, the invention concerns an article of manufacturecomprising a container, a fusion molecule as hereinabove defined withinthe container, and a label or package insert on or associated with thecontainer. The label or package insert preferably comprises instructionsfor the treatment or prevention of an immune disease.

[0048] In a further aspect, the present invention concerns methods forthe treatment and prevention of immune-mediated diseases, where thesubject is administered a fusion polypeptide as described herein. In oneembodiment, the invention concerns a method for the treatment of anautoimmune disease, comprising administering at least once, oralternatively multiple times, an effective amount of at least one fusionmolecule as hereinabove defined to a subject diagnosed with or at riskof developing an autoimmune disease. The subject preferably is a human.The autoimmune disease to be treated or prevented is not limited, but insome embodiments, is preferably selected from rheumatoid arthritis,type-I diabetes mellitus and multiple sclerosis. The fusion molecule ashereinabove defined and used in these treatment methods preferablycontain an autoantigens selected from the group consisting of rheumatoidarthritis autoantigen, multiple sclerosis autoantigen, autoimmune type Idiabetes mellitus autoantigen, and portions thereof. More specificallyby name, examples of autoantigens finding use in the fusion molecule ashereinabove defined include myelin basic protein (MBP), proteolipidprotein, myelin oligodendrocyte glycoprotein, αβ-crystallin,myelin-associated glycoprotein, Po glycoprotein, PMP22, 2′,3′-cyclicnucleotide 3′-phosphohydrolase (CNPase), glutamic acid decarboxylase(GAD), insulin, 64 kD islet cell antigen (IA-2, also termed ICA512),phogrin (IA-2β), type II collagen, human cartilage gp39 (HCgp39), andgp130-RAPS.

[0049] In another aspect, the invention provides a method for theprevention of symptoms resulting from a type I hypersensitivity reactionin a subject receiving immunotherapy, comprising administering at leastone fusion molecule to the subject, wherein the fusion moleculecomprises a first polypeptide sequence capable of specific binding to anative IgG inhibitory receptor comprising an immune receptortyrosine-based inhibitory motif (ITIM), functionally connected to asecond polypeptide sequence capable of binding directly, or indirectlythrough a third polypeptide sequence, to a native IgE receptor (FcεR),wherein the first and second polypeptide sequences are other thanantibody variable regions, and wherein said fusion molecule is notcapable of T cell interaction prior to internalization. The secondpolypeptide sequence in this fusion molecule comprises, alternatively,(a) at least a portion of an autoantigen, (b) an allergen, or (c) atleast a portion of an IgE immunoglobulin heavy chain constant regioncapable of binding to a native IgE receptor (FcεR). In a preferredembodiment, the type I hypersensitivity reaction is an anaphylacticresponse. In preferred embodiments of this method, the type Ihypersensitivity symptoms being prevented comprise an anaphylacticresponse. In other embodiments, the first polypeptide comprises at leasta portion of an IgG immunoglobulin heavy chain constant region, and thethird polypeptide is an IgE class antibody.

[0050] In one aspect of this method of the invention, the immunotherapyreceived by the subject is for the treatment of type Ihypersensitivity-mediated disease or autoimmune disease. In variousembodiments of this method, the fusion molecule is administered to thesubject prior to the subject receiving immunotherapy, co-administered tothe subject during immunotherapy, or administered to the subject afterthe subject receives the immunotherapy.

[0051] In yet another aspect, the invention provides a method for theprevention of a type I hypersensitivity disease in a subject receivingimmunotherapy, comprising administering at least one fusion molecule tothe subject, wherein the fusion molecule comprises a first polypeptidesequence capable of specific binding to a native IgG inhibitory receptorcomprising an immune receptor tyrosine-based inhibitory motif (ITIM),functionally connected to a second polypeptide sequence capable ofbinding directly, or indirectly through a third polypeptide sequence, toa native IgE receptor (FcεR), wherein the first and second polypeptidesequences are other than antibody variable regions, and wherein saidfusion molecule is not capable of T cell interaction prior tointernalization. The second polypeptide sequence in this fusion moleculecomprises, alternatively, (a) at least a portion of an autoantigen, (b)an allergen, or (c) at least a portion of an IgE immunoglobulin heavychain constant region capable of binding to a native IgE receptor(FcεR).

BRIEF DESCRIPTION OF THE FIGURES

[0052]FIG. 1 shows the nucleotide sequence encoding the human IgG₁ heavychain constant region (SEQ ID NO: 1).

[0053]FIG. 2 shows the amino acid sequence of the human IgG₁ heavy chainconstant region (SEQ ID NO: 2). In the sequence, the CH1 domain extendsfrom amino acid position 122 to amino acid position 219, the hingeregion extends from amino acid position 220 to amino acid position 231,the CH2 domain extends from amino acid position 232 to amino acidposition 344, and the CH3 domain extends from amino acid position 345 toamino acid 451 (the C-terminus).

[0054]FIG. 3 shows the amino acid sequence of the hinge-CH2—CH3 portionof the human IgG₁ heavy chain constant region (SEQ ID NO: 3).

[0055]FIG. 4 shows the nucleotide sequence encoding the human IgE heavychain constant region (SEQ ID NO: 4).

[0056]FIG. 5 shows the amino acid sequence of the human IgE heavy chainconstant region (SEQ ID NO: 5).

[0057]FIG. 6 shows the amino acid sequence of the CH2—CH3-CH4 portion ofthe human IgE heavy chain constant region (SEQ ID NO: 6).

[0058]FIG. 7 shows the amino acid sequence of theγhinge-CHγ2-CHγ3-(Gly₄Ser)₃-CHε2-CHε3-CHε3 fusion molecule (GE2) of theinvention (SEQ ID NO: 7).

[0059]FIG. 8 illustrates the dose-dependent inhibition of basophilhistamine release using the fusion protein GE2 (±SEM; n=3 separatedonors, each in duplicate). Purified human blood basophils were acidstripped and then sensitized with humanized anti-NP IgE, labeled as IgE,alone or in the presence of GE2 protein or PS that is a purified humanIgE myeloma protein. One hour later, cells were challenged with NP-BSAand the resulting level of histamine release measured.

[0060]FIG. 9 shows results obtained in the transgenic passive cutaneousanaphylaxis (PCA) model described in the Example. Sites were injectedwith 250 ng of human anti-IgE NP along with the indicated amounts of PS(non-specific human IgE) or GE2 chimeric fusion protein. Four hourslater, the animals were challenged intravenously (IV) with 500 μg ofNP-BSA.

[0061]FIG. 10 illustrates GE2 binding to HMC-1 cells that expressFcγRIIb but not FcεRIa.

[0062]FIG. 11 illustrates GE2 binding to 3D10 cells that express FcεRIabut not FcγRIIb.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0063] I. Definitions

[0064] Unless defined otherwise, technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. One skilled in the artwill recognize many methods and materials similar or equivalent to thosedescribed herein, which could be used in the practice of the presentinvention. Indeed, the present invention is in no way limited to themethods and materials described. For purposes of the present invention,the following terms are defined below.

[0065] The term “functionally connected” with reference to the first andsecond polypeptide sequences included in the fusion molecules herein, isused to indicate that such first and second polypeptide sequences retainthe ability to bind to the respective receptors. Thus, after beingconnected to a second polypeptide sequence, the first polypeptidesequence retains the ability of specific binding to a native IgGinhibitory receptor, such as a low-affinity FcγRIIb receptor. Similarly,after being connected to a first polypeptide sequence, the secondpolypeptide sequence retains the ability of specific binding, directlyor indirectly, i.e. through a third polypeptide sequence, to a nativeIgE receptor, such as a native high-affinity IgE receptor, e.g. nativehuman FcεRI, or a native low-affinity IgE receptor, e.g. FcεRII. As aresult, the fusion molecule, comprising the first and second polypeptidesequences functionally connected to each other, is capable ofcross-linking the respective native receptors, such as, for example,FcγRIIb and FcεRI or FcεRII. In order to achieve a functional connectionbetween the two binding sequences within the fusion molecules of theinvention, it is preferred that they retain the ability to bind to thecorresponding receptor with a binding affinity similar to that of anative immunoglobulin heavy chain or other native polypeptide binding tothat receptor.

[0066] The binding is “specific” when the binding affinity of a moleculefor a binding target, e.g. an IgG or IgE receptor, is significantlyhigher (preferably at least about 2-times, more preferably at leastabout 4-times, most preferably at least about 6-times higher) than thebinding affinity of that molecule to any other known native polypeptide.

[0067] The term “inhibitory receptor” is used in the broadest sense andrefers to a receptor capable of down-regulating a biological responsemediated by another receptor, regardless of the mechanism by which thedown-regulation occurs.

[0068] The terms “receptor comprising an immune receptor tyrosine-basedinhibitory motif (ITIM)” and “ITIM-containing receptor” are used torefer to a receptor containing one or more immune receptortyrosine-based inhibitory motifs, ITIMs. The ITIM motif can be generallyrepresented by the formula Val/Ile-Xaa-PTyr-Xaa-Xaa-Leu/Val (where Xaarepresents any amino acid). ITIM-containing receptors include, withoutlimitation, FcγRIIb, gp49b1/gp91 (Arm et al., J. Biol. Chem.266:15966-73 (1991)), p91/PIR-B (Hayami et al., J. Biol. Chem.272:7320-7 (1997)), LIR1-3, 5, 8, LAIR-1; CD22 (van Rossenberg et al.,J. Biol. Chem. Jan. 4, 2001); CTL-4, CD5, p58/70/140 KIR, PIRB2-5; NKBI,Ly49 A/C/E/F/G, NKG2-A/B, APC-R, CD66, CD72, PD-1, SHPS-1, SIRP-α1, ILT1-5, MIR7, 10, hMIR(HM18), hMIR(HM9), Fas(CD95), TGFβ-R, TNF-R1,IFN-ε-R (α- and β-chains), mast cell function Ag, H2-M, HLA-DM, CD 1, CD1-d, CD46, c-cbl, Pyk2/FADK2, P130 Ca rel prot, PGDF-R, LIF, LIR-R, CIS,SOCS13 and 3, as reviewed in Sinclair NR et al., supra. Ligands for manyof these receptors are also known, such as, e.g. the ligand for CD95 iscalled CD95 ligand, the ligands for CTLA-4 are CD80 and CD86, theligands of IFN-γ receptor is IFN-γ, etc. Ligands for CD22 comprise thebasic binding motif Nau5Ac-a(2,6)-Lac, and are discussed, for example invan Rossenberg et al., 2001, supra.

[0069] The term “IgG inhibitory receptor” is used to define a member ofthe inhibitory receptor superfamily (IRS), now know or hereinafterdiscovered, that is capable of attenuating an FcεR-mediated response,regardless of whether it is mediated via IgE acting through ahigh-affinity IgE receptor, e.g. FcεRI, or a low-affinity IgE receptor,or by another mechanism such as an autoantibody to the FcεR. Theresponse preferably is an IgE-mediated allergic response, such as a typeI (immediate hypersensitivity) reaction but could include autoimmunereactions due to anti-FcεRI α-chain antibodies that have been reportedin about half of the cases of chronic idiopathic urticaria.

[0070] The term “native” or “native sequence” refers to a polypeptidehaving the same amino acid sequence as a polypeptide that occurs innature. In accordance with the present invention, a polypeptide can beconsidered “native” regardless of its source, mode of preparation orstate of purification. Thus, such native sequence polypeptide can beisolated from nature or can be produced by recombinant and/or syntheticmeans. The terms “native” and “native sequence” specifically encompassnaturally-occurring truncated or secreted forms (e.g., an extracellulardomain sequence), naturally-occurring variant forms (e.g., alternativelyspliced forms) and naturally-occurring allelic variants of apolypeptide.

[0071] The terms “native FcγRIIb,” “native sequence FcγRIIb,” “nativelow-affinity IgG inhibitory -receptor FcγRIIb,” and “native sequencelow-affinity IgG inhibitory receptor FcγRIIb” are used interchangeably,and refer to FcγRIIb receptors of any species, including any mammalianspecies, as occurring in nature. Preferably, the mammal is human.FcγRIIb is an isoform of the low-affinity IgG receptor FcγRII containingan immunoreceptor tyrosine-based inhibition motif (ITIM). This receptoris the principal FcγRII species in human peripheral blood basophils andcord blood-derived mast cells. For further details see, for example,Malbec and Fridman, Curr. Top. Microbiol. Immunol. 244:13-27 (1999);Cambier, J. C., Proc. Natl. Acad. Sci. USA 94:5993-5995 (1997); and Ottand Cambier, J. Allergy Clin. Immunol. 106(3):429-440 (2000). FcγRIIbhas three alternatively spliced forms designated FcγRIIb1, FcγRIIb2′,and FcγRIIb2, which differ only in their cytoplasmic domain sequences.All three alternatively spliced isoforms contain two extracellularIg-like loops and a single conserved ITIM motif within their cytoplasmictails, and are specifically included within the definition of FcγRIIb,along with other splice variants that might be identified in the future.

[0072] The terms “native FcεRI,” “native sequence FcεRI,” “nativehigh-affinity IgE receptor FcεRI,” and “native sequence high-affinityIgE receptor FcεRI” are used interchangeably and refer to FcεRIreceptors of any species, including any mammalian species, that occur innature. FcεRI is a member of the multi-subunit immune response receptor(MIRR) family of cell surface receptors that lack intrinsic enzymaticactivity but transduce intracellular signals through association withcytoplasmic tyrosine kinases. For further details see, for example,Kinet, J. P., Annu. Rev. Immunol. 17:931-972 (1999) and Ott and Cambier,J. Allergy Clin. Immunol., 106:429-440 (2000).

[0073] The terms “native FcεRII (CD23),” “native sequence FcεRII(CD23),” native low-affinity IgE receptor FcεRII (CD23),” “nativesequence low-affinity IgE receptor FcεRII (CD23)” are usedinterchangeably and refer to FcεRII (CD23) receptors of any species,including any mammalian species, that occur in nature. Several groupshave cloned and expressed low-affinity IgE receptors of various species.The cloning and expression of a human low-affinity IgE receptor isreported, for example, by Kikutani et al., Cell 47:657-665 (1986), andLudin et al., EMBO J. 6:109-114 (1987). The cloning and expression ofcorresponding mouse receptors is disclosed, for example, by Gollnick etal., J. Immunol. 144:1974-82 (1990), and Kondo et al., Int. Arch.Allergy Immunol. 105:38-48 (1994). The molecular cloning and sequencingof CD23 for horse and cattle has been recently reported by Watson etal., Vet. Immunol. Immunopathol. 73:323-9 (2000). For an earlier reviewof the low-affinity IgE receptor see also Delespesse et al., Immunol.Rev. 125:77-97 (1992).

[0074] The term “mammal” or “mammalian species” refers to any animalclassified as a mammal, including humans, domestic and farm animals, andzoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep,pigs, goats, rabbits, as well as rodents such as mice and rats, etc.Preferably, the mammal is human.

[0075] The terms “subject” or “patient,” as used herein, are usedinterchangeably, and can refer to any to animal, and preferably amammal, that is the subject of an examination, treatment, analysis, testor diagnosis. In one embodiment, humans are a preferred subject. Asubject or patient may or may not have a disease or other pathologicalcondition.

[0076] The terms “peptide,” “polypeptide” and “protein,” in singular orplural, as used herein, all refer to a primary sequence of amino acidsjoined to each other in a linear chain by covalent peptide bonds. Ingeneral, a peptide consists of a small number of amino acid residues,typically from two to about 50 amino acids in length, and is shorterthan a protein. As used in the art, the term “peptides” can be usedinterchangeably with “oligopeptides” and “oligomers.” The term“polypeptide” encompasses peptides and proteins. Peptides, polypeptidesand proteins can be from a natural source, or be recombinant, orsynthetic. Polypeptides, as defined herein, may contain amino acidsother than the 20 naturally occurring amino acids, and may includemodified amino acids. The modification can be anywhere within thepolypeptide molecule, such as, for example, at the terminal amino acids,and may be due to natural processes, such as processing and otherpost-translational modifications, or may result from chemical and/orenzymatic modification techniques which are well known to the art. Theknown modifications include, without limitation, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cystine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. Such modificationsare well known to those of skill and have been described in great detailin the scientific literature, such as, for instance, Creighton, T. E.,Proteins—Structure And Molecular Properties, 2nd Ed., W. H. Freeman andCompany, New York (1993); Wold, F., “Posttranslational ProteinModifications: Perspectives and Prospects,” in PosttranslationalCovalent Modification of Proteins, Johnson, B. C., ed., Academic Press,New York (1983), pp. 1-12; Seifter et al., “Analysis for proteinmodifications and nonprotein cofactors,” Meth. Enzymol. 182:626-646(1990), and Rattan et al., Ann. N.Y Acad. Sci. 663:48-62 (1992).

[0077] Modifications can occur anywhere in a polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. In fact, blockage of the amino or carboxyl group in apolypeptide, or both, by a covalent modification, is common in naturallyoccurring and synthetic polypeptides and such modifications may bepresent in polypeptides of the present invention, as well. For instance,the amino terminal residue of polypeptides made in E. coli, prior toproteolytic processing, almost invariably will be N-formylmethionine.Accordingly, when glycosylation is desired, a polypeptide is expressedin a glycosylating host, generally eukaryotic host cells. Insect cellsoften carry out the same post-translational glycosylations as mammaliancells and, for this reason, insect cell expression systems have beendeveloped to express efficiently mammalian proteins having nativepatterns of glycosylation.

[0078] It will be appreciated that polypeptides are not always entirelylinear. For instance, polypeptides may be branched as a result ofubiquitination, and they may be circular, with or without branching,generally as a result of post-translational events, including naturalprocessing and events brought about by human manipulation which do notoccur naturally. Circular, branched and branched circular polypeptidesmay be synthesized by non-translation natural process and by entirelysynthetic methods, as well. Such structures are within the scope of thepolypeptides as defined herein.

[0079] Amino acids are represented by their common one- or three-lettercodes, as is common practice in the art. Accordingly, the designationsof the twenty naturally occurring amino acids are as follows:Alanine=Ala (A); Arginine=Arg (R); Aspartic Acid=Asp (D); Asparagine-Asn(N); Cysteine=Cys (C); Glutamic Acid=Glu (E); Glutamine=Gln (O);Glycine=Gly (G); Histidine=His (H); Isoleucine=Ile (I); Leucine=Leu (L);Lysine=Lys (K); Methionine=Met (M); Phenylalanine=Phe (F); Proline=Pro(P); Serine=Ser (S); Threonine=Thr (T); Tryptophan=Trp (W); Tyrosine=Tyr(Y); Valine—Val (V). The polypeptides herein may include all L-aminoacids, all D-amino acids or a mixture thereof. The polypeptidescomprised entirely of D-amino acids may be advantageous in that they areexpected to be resistant to proteases naturally found within the humanbody, and may have longer half-lives.

[0080] The term “amino acid sequence variant” refers to molecules withsome differences in their amino acid sequences as compared to areference (e.g. native sequence) polypeptide. The amino acid alterationsmay be substitutions, insertions, deletions or any desired combinationsof such changes in a native amino acid sequence.

[0081] Substitutional variants are those that have at least one aminoacid residue in a native sequence removed and a different amino acidinserted in its place at the same position. The substitutions may besingle, where only one amino acid in the molecule has been substituted,or they may be multiple, where two or more amino acids have beensubstituted in the same molecule.

[0082] Insertional variants are those with one or more amino acidsinserted immediately adjacent to an amino acid at a particular positionin a native amino acid sequence. Immediately adjacent to an amino acidmeans connected to either the α-carboxy or α-amino functional group ofthe amino acid.

[0083] Deletional variants are those with one or more amino acids in thenative amino acid sequence removed. Ordinarily, deletional variants willhave at least one amino acid deleted in a particular region of themolecule.

[0084] The term “sequence identity” is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in a reference polypeptide sequence (e.g., a nativepolypeptide sequence), after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity,and not considering any “conservative substitutions” as part of thesequence identity, wherein conservative amino acid substitutions are thesubstitution of one amino acid for a different amino acid having similarchemical properties. The % sequence identity values are generated by theNCBI BLAST2.0 software as defined by Altschul et al., (1997), “GappedBLAST and PSI-BLAST: a new generation of protein database searchprograms”, Nucleic Acids Res., 25:3389-3402. The parameters are set todefault values, with the exception of the Penalty for mismatch, which isset to −1.

[0085] The term “sequence similarity” as used herein, is the measure ofamino acid sequence identity, as described above, and in addition alsoincorporates conservative amino acid substitutions.

[0086] “Stringent” hybridization conditions are sequence dependent andwill be different with different environmental parameters (e.g., saltconcentrations, and presence of organics). Generally, stringentconditions are selected to be about 5° C. to 20° C. lower than thethermal melting point (T_(m)) for the specific nucleic acid sequence ata defined ionic strength and pH. Preferably, stringent conditions areabout 5° C. to 10° C. lower than the thermal melting point for aspecific nucleic acid bound to a perfectly complementary nucleic acid.The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of a nucleic acid (e.g., tag nucleic acid) hybridizes to aperfectly matched probe.

[0087] “Stringent” wash conditions are ordinarily determined empiricallyfor hybridization of each set of tags to a corresponding probe array.The arrays are first hybridized (typically under stringent hybridizationconditions) and then washed with buffers containing successively lowerconcentrations of salts, or higher concentrations of detergents, or atincreasing temperatures until the signal to noise ratio for specific tonon-specific hybridization is high enough to facilitate detection ofspecific hybridization. Stringent temperature conditions will usuallyinclude temperatures in excess of about 30° C., more usually in excessof about 37° C., and occasionally in excess of about 45° C. Stringentsalt conditions will ordinarily be less than about 1000 mM, usually lessthan about 500 mM, more usually less than about 400 mM, typically lessthan about 300 mM, preferably less than about 200 mM, and morepreferably less than about 150 mM. However, the combination ofparameters is more important than the measure of any single parameter.See, e.g., Wetmur et al., J. Mol. Biol. 31:349-70 (1966), and Wetmur,Critical Reviews in Biochemistry and Molecular Biology 26(34):227-59(1991).

[0088] In a preferred embodiment, “stringent conditions” or “highstringency conditions,” as defined herein, may be hybridization in 50%formamide, 6× SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution,sonicated salmon sperm DNA (100 μg/ml), 0.5% SDS, and 10% dextransulfate at 42° C., with washes at 42° C. in 2× SSC (sodiumchloride/sodium citrate) and 0.1% SDS at 55° C., followed by ahigh-stringency wash consisting of 0.2× SSC containing 0.1% SDS at 42°C.

[0089] The terms “complement,” “complementarity” or “complementary,” asused herein, are used to describe single-stranded polynucleotidesrelated by the rules of antiparallel base-pairing. For example, thesequence 5′-CTAGT-3′ is completely complementary to the sequence5′-ACTAG-3′. Complementarity may be “partial,” where the base pairing isless than 100%, or complementarity may be “complete” or “total,”implying perfect 100% antiparallel complementation between the twopolynucleotides. By convention in the art, single-stranded nucleic acidmolecules are written with their 5′ ends to the left, and their 3′ endsto the right.

[0090] The term “immunoglobulin” (Ig) is used to refer to theimmunity-conferring portion of the globulin proteins of serum, and toother glycoproteins, which may not occur in nature but have the samefunctional characteristics. The term “immunoglobulin” or “Ig”specifically includes “antibodies” (Abs). While antibodies exhibitbinding specificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules that lack antigenspecificity. Native immunoglobulins are secreted by differentiated Bcells termed plasma cells, and immunoglobulins with unidentified antigenspecificity are constitutively produced at low levels by the immunesystem and at increased levels by myelomas. As used herein, the terms“immunoglobulin,” “Ig,” and grammatical variants thereof are used toinclude antibodies, and Ig molecules without known antigen specificity,or without antigen binding regions.

[0091] The term “specific antibody” as used herein is intended toindicate an antibody that has binding specificity to a specifiedantigen. Although all antibodies are by nature specific for at least oneepitope, the expression “specific antibody” implies that the antibodybinds specifically to a particular known antigen. Binding specificity isdetermined by the amino acid sequences and conformation of the Igvariable domains of the heavy and light chains, as well as theconformation of the recognized epitope. The antigenic epitopestypically, but not exclusively, consist of small amino acid sequencedomains. For example, the anti-myelin-basic-protein (MBP) autoantibodyis specific for the MBP antigen, and more specifically, for the MBP₈₃₋₉₉region. “Specific binding” and “specifically binding” refer to theinteraction between an antibody and its specific antigen that isdependent on the presence of complementary structures on the antigenicepitope and the antibody.

[0092] Native immunoglobulins are usually heterotetrameric glycoproteinsof about 150,000 daltons, composed of two identical light (L) chains andtwo identical heavy (H) chains. Each light chain is linked to a heavychain by one covalent disulfide bond, while the number of disulfidelinkages varies among the heavy chains of different immunoglobulinisotypes. Each heavy and light chain also has regularly spacedintrachain disulfide bridges. Each heavy chain has at one end a variabledomain (V_(H)) followed by a number of constant domains. Each lightchain has a variable domain at one end (V_(L)) and a constant domain atits other end; the constant domain of the light chain is aligned withthe first constant domain of the heavy chain, and the light-chainvariable domain is aligned with the variable domain of the heavy chain.Particular amino acid residues are believed to form an interface betweenthe light- and heavy-chain variable domains.

[0093] The main Ig isotypes (classes) found in serum, and thecorresponding Ig heavy chains, shown in parentheses, are listed below:

[0094] IgG (γ chain): the principal Ig in serum, the main antibodyraised in response to an antigen, has four major subtypes, several ofwhich cross the placenta;

[0095] IgE (ε chain): this Ig binds tightly to mast cells and basophils,and when additionally bound to antigen, causes release of histamine andother mediators of immediate hypersensitivity; plays a primary role inallergic reactions, including hay fever, asthma and anaphylaxis; and mayserve a protective role against parasites;

[0096] IgA (α chain): this Ig is present in external secretions, such assaliva, tears, mucous, and colostrum;

[0097] IgM (μ chain): the Ig first induced in response to an antigen; ithas lower affinity than antibodies produced later and is pentameric; and

[0098] IgD (δ chain): this Ig is found in relatively high concentrationsin umbilical cord blood, serves primarily as an early cell receptor forantigen, and is the main lymphocyte cell surface molecule.

[0099] Antibodies of the IgG, IgE, IgA, IgM, and IgD isotypes may havethe same variable regions, i.e. the same antigen binding cavities, eventhough they differ in the constant region of their heavy chains. Theconstant regions of an immunoglobulin, e.g. antibody are not involveddirectly in binding the antibody to an antigen, but correlate with thedifferent effector functions mediated by antibodies, such as complementactivation or binding to one or more of the antibody Fc receptorsexpressed on basophils, mast cells, lymphocytes, monocytes andgranulocytes.

[0100] Some of the main antibody isotypes (classes) are divided intofurther sub-classes. IgG has four known subclasses: IgG₁ (γ₁), IgG₂(γ₂), IgG₃ (γ₃), and IgG₄ (γ₄), while IgA has two known sub-classes:IgA₁ (α₁) and IgA₂ (α₂).

[0101] A light chain of an Ig molecule is either a κ or a λ chain.

[0102] The constant region of an immunoglobulin heavy chain is furtherdivided into globular, structurally discrete domains, termed heavy chainconstant domains. For example, the constant region of an IgG₁immunoglobulin heavy chain comprises three constant domains, CH1, CH2and CH3, and a hinge region between the CH1 and CH2 domains. The IgEimmunoglobulin heavy chain comprises four constant domains: CH1, CH2,CH3 and CH4 and does not have a hinge region.

[0103] Immunoglobulin sequences, including sequences of immunoglobulinheavy chain constant regions are well known in the art and aredisclosed, for example, in Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitute of Health, Bethesda, Md. (1991). For a discussion of the humanIgG₁ heavy chain constant region (≡₁), see also Ellison et al., Nucl.Acid Res. 10:4071-4079 (1982); and Takahashi et al., Cell 29:671-679(1982). For a discussion of the human IgG₂ constant region (γ₂), seealso Krawinkel et al., EMBO J. 1:403-407 (1982); Ellison et al., Proc.Nat. Acad. Sci. USA 79:1984-1988 (1982); and Takahashi et al. (1982),supra. For a discussion of human IgG₃ heavy chain constant region (73),see also Krawinkel et al., (1982), supra, and Takahashi et al. (1982),supra. For a discussion of human IgG₄ heavy chain constant region (γ4),see also Ellison et al., DNA 1:1 1-18 (1982), Krawinkel et al. (1982),supra, and Takahashi et al. (1982), supra. For a discussion of the humanIgE heavy chain constant region (ε), see also Max et al., Cell29:691-699 (1982). IgE isoforms are described in Saxon et al., J.Immunol. 147:4000 (1991); Peng et al., J. Immunol. 148:129-136 (1992);Zhang et al., J. Exp. Med. 176:233-243 (1992); and Hellman, Eur. J.Immunol. 23:159-167 (1992).

[0104] The term “antigen,” as used herein, refers to any agent that isrecognized by an antibody, while the term “immunogen” refers to anyagent that can elicit an immunological response in a subject. The terms“antigen” and “immunogen” both encompass, but are not limited to,polypeptides. In most, but not all cases, antigens are also immunogens.The term “allergen,” and grammatical variants thereof, as used herein,refer to antigens that are capable of inducing IgE-mediated responses,e.g., allergies. An allergen can be almost anything that acts as anantigen and stimulates an IgE-mediated allergic reaction. Commonallergens can be found, for example, in food, pollen, mold, house dustwhich may contain mites as well as dander from house pets, venom frominsects such as bees, wasps and mosquitoes.

[0105] The terms “epitope” or “antigenic determinant” as used herein,refer to that portion of an antigen that makes contact with a particularantibody variable region, and thus imparts specificity to theantigen/antibody binding. A single antigen may have more than oneepitope. An immunodominant epitope is an epitope on an antigen that ispreferentially recognized by antibodies to the antigen. In some cases,where the antigen is a protein, the epitope can be “mapped,” and an“antigenic peptide” produced corresponding approximately to just thoseamino acids in the protein that are responsible for the antibody/antigenspecificity. Such “antigenic peptides” find use in peptideimmunotherapies.

[0106] The terms “autoantigen” and “self antigen” and grammaticalequivalents, as used herein, refer to an antigen endogenous to anindividual's physiology, that is recognized by either the cellularcomponent (T-cell receptors) or humoral component (antibodies) of thatindividual's immune system. The presence of autoantigens, andconsequently autoantibodies and/or self-reactive T-cells, is frequently,but not absolutely, associated with disease states. Autoantibodies maybe detected in disease-free individuals. Autoantigens are frequently,but not exclusively, polypeptides. An understanding of the mechanismsunderlying the recognition of autoantigens, the loss of normalself-recognition, or the mechanisms inducing autoimmunity are notnecessary to make or use the present invention.

[0107] The term “autoantibody,” as used herein, is intended to refer toany antibody produced by a host organism that binds specifically to anautoantigen, as defined above. The presence of autoantibodies and/orself-reactive T-cells is referred to herein as “autoimmunity.” Thepresence of autoantibodies or self-reactive T-cells in a subject isfrequently, but not absolutely, associated with disease (i.e.,autoimmune disease).

[0108] The terms “disease,” “disorder” and “condition” are usedinterchangeably herein, and refer to any disruption of normal bodyfunction, or the appearance of any type of pathology. The etiologicalagent causing the disruption of normal physiology may or may not beknown. Furthermore, although two patients may be diagnosed with the samedisorder, the particular symptoms displayed by those individuals may ormay not be identical.

[0109] The terms “autoimmune disease,” “autoimmune condition” or“autoimmune disorder,” as used interchangeably herein, refer to a set ofsustained organ-specific or systemic clinical symptoms and signsassociated with altered immune homeostasis that is manifested byqualitative and/or quantitative defects of expressed autoimmunerepertoires. Autoimmune disease pathology is manifested as a result ofeither structural or functional damage induced by the autoimmuneresponse. Autoimmune diseases are characterized by humoral (e.g.,antibody-mediated), cellular (e.g., cytotoxic T lymphocyte-mediated), ora combination of both types of immune responses to epitopes onself-antigens. The immune system of the affected individual activatesinflammatory cascades aimed at cells and tissues presenting thosespecific self-antigens. The destruction of the antigen, tissue, celltype or organ attacked gives rise to the symptoms of the disease. Theautoantigens are known for some, but not all, autoimmune diseases.

[0110] The terms “immunotherapy,” “desensitisation therapy,”“hyposensitisation therapy,” “tolerance therapy” and the like, as usedherein, describe methods for the treatment of various hypersensitivitydisorders, where the avoidance of an allergen or autoantigen is notpossible or is impractical. As used herein, these terms are used largelyinterchangeably. These methods generally entail the delivery to asubject of the antigenic material in a controlled manner to inducetolerance to the antigen and/or downregulate an immune response thatoccurs upon environmental exposure to the antigen. These therapiestypically entail injections of the antigen (e.g., an allergen orautoantigen) over an extended period of time (months or years) ingradually increasing doses. The antigen used in the immunotherapies istypically, but not exclusively, polypeptides. For example, hayfeverdesensitisation therapy downregulates allergic response to airbornpollen, where the subject is injected with a pollen extract. From aclinical perspective, these treatments are suboptimal, as the injectionsare typically painful, as well as inconvenient. Furthermore, asignificant risk of potentially life-threatening anaphylactic responsesduring the therapies exists. Adapting immunotherapy techniques for thetreatment of various autoimmune disorders has been proposed, where theautoantigen is administered to a subject in the hope of inducingtolerance to the autoantigen, and thereby eliminating the immunedestruction of the endogenous autoantigen or autoantigenic tissue. Forexample, insulin and myelin-basic-protein have been delivered to animalmodels and humans for the purpose of downregulating autoimmune type-Idiabetes mellitus and multiple sclerosis, respectively.

[0111] The terms “peptide therapy” and “peptide immunotherapy,” and thelike, as used herein, describe methods of immunotherapy, wherein theantigen (e.g., an allergen or autoantigen) delivered to a subject is ashort polypeptide (i.e., a peptide). Furthermore, the peptide deliveredduring peptide therapy may preferably contain only those amino acidsdefining an immunodominant epitope (e.g., the myelin-basic-proteinepitope (MBP83-99).

[0112] The terms “vaccine therapy,” “vaccination” and “vaccinationtherapy,” as used interchangeably herein, refer in general to any methodresulting in immunological prophylaxis. In one aspect, vaccine therapyinduces an immune response, and thus long-acting immunity, to a specificantigen. These methods generally entail the delivery to a subject of animmunogenic material to induce immunity. In this case, the immunogenicmaterial is generally killed microbes of virulent stains or living,attenuated strains, or derivatives or products of virulent pathogens. Inanother aspect, the “vaccine therapy” refers to a method for thedownregulation of an immune potential to a particular antigen (e.g., tosuppress an allergic response). This type of vaccine therapy is alsoreferred to as “tolerance therapy.” Vaccine therapies typically entail aseries of parenteral or oral administrations of the immunogenic materialover an extended period of time.

[0113] The terms “fragment,” “portion” and “part,” as usedinterchangeably herein, refer to any composition of matter that issmaller than the whole of the composition of matter from which it isderived. For example, a portion of a polypeptide may range in size fromtwo amino acid residues to the entire amino acid sequence minus oneamino acid. However, in most cases, it is desirable for a “portion” or“fragment” to retain an activity or quality which is essential for itsintended use. For example, useful portions of an antigen are thoseportions that retain an epitope determinant. Also, in one embodiment,useful portions of an immunoglobulin heavy chain constant region arethose portions that retain the ability to form covalent homodimericstructures and are able to bind an Fcγ receptor.

[0114] The term “at least a portion,” as used herein, is intended toencompass portions as well as the whole of the composition of matter.

[0115] The terms “type I allergic reaction,” “immediatehypersensitivity,” “atopic allergy,” “type-I hypersensitivity,” and thelike, as used herein, refer to the physiological response that occurswhen an antigen entering the body encounters mast cells or basophilswhich have been sensitized by IgE attached to its high-affinityreceptor, FcεRI on these cells. When an allergen reaches the sensitizedmast cell or basophil, it cross-links surface-bound IgE, causing anincrease in intracellular calcium (Ca²⁺) that triggers the release ofpre-formed mediators, such as histamine and proteases, and newlysynthesized, lipid-derived mediators such as leukotrienes andprostaglandins. These autocoids produce the clinical symptoms ofallergy. In addition, cytokines, e.g., IL-4, TNF-alpha, are releasedfrom degranulating basophils and mast cells, and serve to augment theinflammatory response that accompanies an IgE reaction (see, e.g.,Immunology, Fifth Edition, Roitt et al., eds., 1998, pp. 302-317). Thespecific manifestations of the hypersensitivity reaction in thesensitive or allergic subject depends on the site of the allergenexposure, the dose of allergen exposure, the reactivity of the organs inthe subject (e.g., over-reactive lungs or nose) and the full panoply ofthe immune response to the allergen in that subject.

[0116] Symptoms and signs associated with type I hypersensitivityresponses are extremely varied due to the wide range of tissues andorgans that can be involved. These symptoms and signs can include, butare not limited to: itching of the skin, eyes, and throat, swelling andrashes of the skin (angioedema and urticaria/hives), hoarseness anddifficulty breathing due to swelling of the vocal cord area, apersistent bumpy red rash that may occur anywhere on the body, shortnessof breath and wheezing (from tightening of the muscles in the airwaysand plugging of the airways, i.e., bronchoconstriction) in addition toincreased mucus and fluid production, chest tightness and pain due toconstruction of the airway muscles, nausea, vomiting diarrhea, dizzinessand fainting from low blood pressure, a rapid or irregular heartbeat andeven death as a result of airway and/or cardiac compromise.

[0117] Examples of disease states that result from allergic reactions,and demonstrating hypersensitivity symptoms and/or signs include, butare not limited to, allergic rhinitis, allergic conjunctivitis, atopicdermatitis, allergic [extrinsic] asthma, some cases of urticaria andangioedema, food allergy, and anaphylactic shock in which there issystemic generalized reactivity and loss of blood pressure that may befatal.

[0118] The terms “anaphylaxis,” “anaphylactic response,” “anaphylacticreaction,” “anaphylactic shock,” and the like, as used interchangeablyherein, describe the acute, often explosive, IgE-mediated systemicphysiological reaction that occurs in a previously sensitized subjectwho receives the sensitizing antigen. Anaphylaxis occurs when thepreviously sensitizing antigen reaches the circulation. When the antigenreacts with IgE on basophils and mast cells, histamine, leukotrienes,and other inflammatory mediators are released. These mediators cause thesmooth muscle contraction (responsible for wheezing and gastrointestinalsymptoms) and vascular dilation (responsible for the low blood pressure)that characterize anaphylaxis. Vasodilation and escape of plasma intothe tissues causes urticaria and angioedema and results in a decrease ineffective plasma volume, which is the major cause of shock. Fluidescapes into the lung alveoli and may produce pulmonary edema.Obstructive angioedema of the upper airway may also occur. Arrhythmiasand cardiogenic shock may develop if the reaction is prolonged. The term“anaphylactoid reaction” refers to a physiological response thatdisplays characteristics of an anaphylactic response.

[0119] Symptoms of an anaphylactic reaction vary considerably amongpatients. Typically, in about 1 to 15 minutes (but rarely after as longas 2 hours), symptoms can include agitation and flushing, palpitations,paresthesias, pruritus, throbbing in the ears, coughing, sneezing,urticaria and angioedema, vasodilation, and difficulty breathing owingto laryngeal edema or bronchospasm. Nausea, vomiting, abdominal pain,and diarrhea are also sometimes observed. Shock may develop withinanother 1 or 2 minutes, and the patient may convulse, becomeincontinent, unresponsive, and succumb to cardiac arrest, massiveangioedema, hypovolemia, severe hypotension and vasomotor collapse andprimary cardiovascular collapse. Death may ensue at this point if theantagonist epinephrine is not immediately available. Mild forms ofanaphylactic response result in various symptoms including generalizedpruritus, urticaria, angioedema, mild wheezing, nausea and vomiting.Patients with the greatest risk of anaphylaxis are those who havereacted previously to a particular drug or antigen.

[0120] The terms “vector”, “polynucleotide vector”, “construct” and“polynucleotide construct” are used interchangeably herein. Apolynucleotide vector of this invention may be in any of several forms,including, but not limited to, RNA, DNA, RNA encapsulated in aretroviral coat, DNA encapsulated in an adenovirus coat, DNA packaged inanother viral or viral-like form (such as herpes simplex, andadeno-associated virus (AAV)), DNA encapsulated in liposomes, DNAcomplexed with polylysine, complexed with synthetic polycationicmolecules, conjugated with transferrin, complexed with compounds such aspolyethylene glycol (PEG) to immunologically “mask” the molecule and/orincrease half-life, or conjugated to a non-viral protein. Preferably,the polynucleotide is DNA. As used herein, “DNA” includes not only basesA, T, C, and G, but also includes any of their analogs or modified formsof these bases, such as methylated nucleotides, internucleotidemodifications such as uncharged linkages and thioates, use of sugaranalogs, and modified and/or alternative backbone structures, such aspolyamides.

[0121] A “host cell” includes an individual cell or cell culture whichcan be or has been a recipient of any vector of this invention. Hostcells include progeny of a single host cell, and the progeny may notnecessarily be completely identical (in morphology or in total DNAcomplement) to the original parent cell due to natural, accidental, ordeliberate mutation and/or change. A host cell includes cellstransfected or infected in vivo with a vector comprising a nucleic acidof the present invention.

[0122] The term “promoter” means a nucleotide sequence that, whenoperably linked to a DNA sequence of interest, promotes transcription ofthat DNA sequence.

[0123] Nucleic acid is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. For example,DNA for a presequence or secretory leader is operably linked to DNA fora polypeptide if it is expressed as a preprotein that participates inthe secretion of the polypeptide; a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thesequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to facilitate translation. Generally,“operably linked” means that the DNA sequences being linked arecontiguous and, in the case of a secretory leader, contiguous and inreading phase. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites. If suchsites do not exist, the synthetic oligonucleotide adaptors or linkersare used in accord with conventional practice.

[0124] The term “IgE-mediated biological response” is used to refer to acondition or disease which is characterized by signal transductionthrough an IgE receptor, including the high-affinity IgE receptor,FcεRI, and the low-affinity IgE receptor FcεRII. The definitionincludes, without limitation, conditions associated with anaphylactichypersensitivity and atopic allergies, such as, for example, asthma,allergic rhinitis, atopic dermatitis, food allergies, chronic urticariaand angioedema, as well as the serious physiological condition ofanaphylactic shock, usually caused by bee stings or medications such aspenicillin.

[0125] The terms “treat” or “treatment” refer to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to prevent or slow down (lessen) an undesired physiological change ordisorder. For purposes of this invention, beneficial or desired clinicalresults include, but are not limited to, alleviation of symptoms,diminishment of extent of disease, stabilized (i.e., not worsening)state of disease, delay or slowing of disease progression, ameliorationor palliation of the disease state, and remission (whether partial ortotal), whether detectable or undetectable. Those in need of treatmentinclude those already with the condition or disorder as well as thoseprone to have the condition or disorder or those in which the conditionor disorder is to be prevented.

[0126] “Chronic” administration refers to administration of the agent(s)in a continuous mode as opposed to an acute mode, so as to maintain adesired effect or level of agent(s) for an extended period of time.

[0127] “Intermittent” administration is treatment that is notconsecutively done without interruption, but rather is periodic innature.

[0128] Administration “in combination with” one or more furthertherapeutic agents includes simultaneous (concurrent) and consecutiveadministration in any order.

[0129] An “effective amount” is an amount sufficient to effectbeneficial or desired therapeutic (including preventative) results. Aneffective amount can be administered in one or more administrations.

[0130] “Carriers” as used herein include pharmaceutically acceptablecarriers, excipients, or stabilizers which are nontoxic to the cell ormammal being exposed thereto at the dosages and concentrations employed.Often the physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0131] The terms “protease,” “peptidase” or “proteinase,” andgrammatical equivalents as used interchangeably herein, refer to anypolypeptide that is able to cleave covalent peptide bonds. Collectively,these proteases, peptidases and proteinases can be referred to as“proteolytic enzymes.”Numerous proteolytic enzymes are known, and aregenerally classified by their cleavage specificities, or lack thereof.Cleavage specificity can be determined by the primary sequence of aminoacids in the target polypeptide, as well as the spatial conformation ofthose amino acids. For example, exopeptidase proteolytic activitycleaves either an amino-terminal (N-terminal) amino acid, or thecarboxy-terminal (C-terminal) amino acid from a larger polypeptide.Endopeptidase enzymes cleave at a peptide bond that is internal to thepolypeptide (i.e., not at either the N-terminal or C-terminal amino acidpositions). Some proteolytic enzymes have very fastidious cleavagespecificity, where cleavage requires recognition of an extended aminoacid target sequence. Alternatively, some peptidases have a more relaxedrequirement for cleavage site recognition, and require only the presenceof a single amino acid to target the proteolysis event. For example,cysteine, aspartate or arginine family endoproteases will cleave atinternal cysteine, aspartate or asparagine amino acid residues,respectively. In some cases, the cysteine, aspartate or arginineendoprotease will require the presence or absence of other amino acidsadjacent to or in the vicinity of the target cysteine, aspartate orarginine residue to effect cleavage. For example, some aspartate familyendopeptidases are unable to cleave the aspartate peptide bond if theadjacent amino acid is a proline. Thus, a peptidase “cleavage site,” asused herein, may encompass more amino acids than only the target residuefor cleavage.

[0132] II. Description of Certain Preferred Embodiments

[0133] 1. Design of the Fusion Molecules

[0134] In one embodiment, the present invention provides fusionmolecules that are capable of attenuating a biological response mediatedby an FcεR, such as conditions associated with anaphylactichypersensitivity (including anaphylactic reactions resulting frompeptide therapies for the treatment of allergic or autoimmune diseases)and atopic allergies, by cross-linking an inhibitory receptor expressedon mast cells and/or basophils with an IgE receptor. The actual sequenceof the fusion molecule will depend on the targeted inhibitory receptor,such as an ITIM-containing receptor, e.g. various forms of FcγRIIb,inhibitory members of the gp49 family, especially gp49b1, p91/PIR-B,LAIR-1, LIR-1, or CD22, and on the targeted IgE receptors, e.g. FcεR1 orFcεR11.

[0135] In a preferred embodiment, the inhibitory receptor is a nativelow-affinity FcγRIIb receptor, and the IgE receptor is a nativehigh-affinity or low-affinity IgE receptor, i.e. FcγRI or FcεRII, morepreferably FcεRI. Accordingly, the first polypeptide sequence present inthe fusion molecules binds to the native low-affinity FcγRIIb receptor,while the second polypeptide sequence, which is functionally connectedto the first polypeptide sequence, binds to a native FcεRI or FcεRII,preferably FcεRI. When the goal is to cross-link a native FcγRIIbreceptor with a native FcεRI receptor by direct binding of the first andsecond polypeptide sequences present in the single-chain fusionmolecules of the invention to the respective receptors, the first andsecond polypeptide sequences, which are functionally connected, arepreferably, but not necessarily, designed to bind to the respectivereceptors at essentially the same region(s) as native IgG and IgE,respectively. It has been reported that the CH2-CH3 interface of the IgGFc domain contains the binding sites for a number of Fc receptors,including the FcγRIIb low-affinity receptor (Wines et al., J. Immunol.164(10):5313-5318 (2000)). Based on FcεRI binding studies, Presta etal., J. Biol. Chem. 269:26368-26373 (1994) proposed that six amino acidresidues (Arg-408, Ser-41 1, Lys-415, Glu-452, Arg-465, and Met-469)located in three loops, C-D, E-F, and F-G, computed to form the outerridge on the most exposed side of the human IgE heavy chain CH3 domain,are involved in binding to the high-affinity receptor FcεRI, mostly byelectrostatic interactions. Helm et al., J. Cell Biol. 271(13):7494-7500(1996), reported that the high-affinity receptor binding site in the IgEmolecule includes the Pro343-Ser353 peptide sequence within the CH3domain of the IgE heavy chain, but sequences N- or C-terminal to thiscore peptide are also necessary to provide structural scaffolding forthe maintenance of a receptor binding conformation. In particular, theyfound that residues, including His, in the C-terminal region of theε-chain make an important contribution toward the maintenance of thehigh-affinity of interaction between IgE and FcεRI. The first and secondpolypeptide sequences within the fusion molecules of the invention arepreferably designed to bind to residues within such binding regions.

[0136] In another class of the fusion molecules of the invention, thefirst polypeptide sequence will bind to an ITIM-containing receptor,other than FcγRIIb, expressed on mast cells, basophils and/or B cells.For example, the first polypeptide sequence may contain a region capableof specific binding to an inhibitory member of the gp49 family, such asgp49b1, which is a member of the immunoglobulin superfamily, ispreferentially expressed on mast cells and mononuclear macrophages, andcontains two ITIM motifs in its cytoplasmic domain. AnotherITIM-containing inhibitory receptor is p91, also referred to as PIR-B,which is known to be expressed on B cells and myeloid lineage cells.Further ITIM-containing receptors that might be targeted by the fusionmolecules of the invention include, without limitation, LAIR-1,expressed on B cells, in addition to NK cells, T cells and monocytes;LIR-1, expressed on B cells and monocytes; and CD22 expressed on Bcells. For review of ITIM-containing receptors and related art see, e.g.Mustelin et al., Front. Biosci. 3:d1060-1096 (1998), and Sinclair etal., 1999, supra.

[0137] A second class of fusion molecules of the invention comprise afirst and a second polypeptide sequence, wherein the second polypeptidesequence comprises part or whole of a native allergen or autoantigenamino acid sequence, or a variant thereof, binding between the secondpolypeptide sequence and an IgE receptor occurs indirectly via specificIgE molecules. The allergen- or autoantigen-derived sequence will bindto a specific IgE molecule bound to a high-affinity IgE receptor (FcεRI)on mast cells or basophils and/or to a low-affinity IgE receptor(FcεRII, CD23) on B lymphocytes. The first, inhibitory receptor-binding,sequence is designed as discussed above. In a preferred embodiment, theallergen or autoantigen part of the molecule is a fragment that containsonly a single IgE binding site (or single immunodominant epitope), inorder to avoid antigen cross-linking of IgE on the mast cell surface.

[0138] In a preferred embodiment, the first polypeptide sequence presentin the fusion molecules of the invention has at least about 80%, morepreferably at least about 85%, even more preferably at least about 90%,yet more preferably at least about 95%, most preferably at least about99% sequence identity with the amino acid sequence of the hinge-CH2—CH3region of a native IgG, e.g. IgG, immunoglobulin, preferably nativehuman IgG₁. In a particularly preferred embodiment, the sequenceidentity is defined with reference to the human γhinge-CHγ2-CHγ3sequence of SEQ ID NO: 3.

[0139] In another preferred embodiment, the first polypeptide sequencepresent in the fusion molecules of the invention has at least about 80%,more preferably at least about 85%, even more preferably at least about90%, yet more preferably at least about 95%, most preferably at leastabout 99% sequence identity with the amino acid sequence of a nativeligand of another ITIM-containing receptor expressed on mast cells,basophils and/or B cells, such as gp49b 1 or p91/PIR-B (a cytoplasmicsignaling protein activated by IFN-α, IFN-γ, and IL-6), or mast cellfunction Ag.

[0140] In yet another preferred embodiment, the first polypeptidesequence present in the fusion molecules of the invention has at leastabout 80%, more preferably at least about 85%, even more preferably atleast about 90%, yet more preferably at least about 95%, most preferablyat least about 99% sequence identity with the amino acid sequence ofc-Kit (see, e.g., Yarden et al., EMBO J., 6:3341-3351 [1987]).

[0141] In one embodiment, the second polypeptide sequence present in thefusion molecules of the invention preferably has at least about 80%,more preferably at least about 85%, even more preferably at least about90%, yet more preferably at least about 95%, most preferably at leastabout 99% sequence identity with the amino acid sequence of theCH2-CH3-CH4 region of a native IgE immunoglobulin, preferably nativehuman IgE, or with the sequence of a native allergen or autoantigenprotein. In a particularly preferred embodiment, the sequence identityis defined with reference to the human CHε2-CHε3-CHε4 sequence of SEQ IDNO: 6 or with regard to one of the allergen sequences listed in Table 1below, or, in one preferred embodiment, one of two Ara h2 clones,represented by SEQ ID NOs: 10 and 11, respectively. TABLE 1 SWISS-SWISS-PROT PROT Allergen Entry Accession No. Protein Name Source Aln g 1MPAG_ALNGL P38948 Major Pollen Allergen Pollen of Alnus Aln g 1glutinosa (Alder) Alt a 6 RLA2_ALTAL P42037 60S Acidic RibosomalAlternaria alternata Protein P2 Alt a 7 ALA7_ALTAL P42058 Minor AllergenAlt a 7 Alternaria alternata Alt a 10 DHAL_ALTAL P42041 AldehydeAlternaria alternata Dehydrogenase Alt a 12 RLA1_ALTAL P49148 60S AcidicRibosomal Alternaria alternata Protein P1 Amb a 1 MP11_AMBAR P27759Pollen Allergen Amb a Ambrosia artemisiifolia 1.1 [Precursor] (Shortragweed) Amb a 1 MP12_AMBAR P27760 Pollen Allergen Amb a Ambrosiaartemisiifolia 1.2 [Precursor] (Short ragweed) Amb a 1 MP13_AMBAR P27761Pollen Allergen Amb a Ambrosia artemisiifolia 1.3 [Precursor] (Shortragweed) Amb a 1 MP14_AMBAR P28744 Pollen Allergen Amb a Ambrosiaartemisiifolia 1.4 [Precursor] (Short ragweed) Amb a 2 MPA2_AMBAR P27762Pollen Allergen Amb a Ambrosia artemisiifolia 2 [Precursor] (Shortragweed) Amb a 3 MPA3_AMBEL P00304 Pollen Allergen Amb a Ambrosiaartemisiifolia 3 var. elatior (Short ragweed) Amb a 5 MPA5_AMBEL P02878Pollen Allergen Amb a Ambrosia artemisiifolia 5 var. elatior (Shortragweed) Amb p 5 MPA5_AMBPS P43174 Pollen Allergen Amb p Ambrosiapsilostachya 5-a [Precursor] (Western ragweed) Amb p 5 MP5B_AMBPS P43175Pollen Allergen Amb p Ambrosia psilostachya 5b [Precursor] (Westernragweed) Amb t 5 MPT5_AMBTR P10414 Pollen Allergen Amb t Ambrosiatrifida (Giant 5 [Precursor] ragweed) Api g 1 MPAG_APIGR P49372 MajorAllergen Api g 1 Apium grayeolens (Celery) Api m 1 PA2_APIME P00630Phospholipase A2 Apis mellifera [Precursor] [Fragment] (Honeybee) Api m2 HUGA_APIME Q08169 Hyaluronoglucosamin- Apis mellifera idase[Precursor] (Honeybee) Api m 3 MEL_APIME P01501 Melittin [Precursor]Apis mellifera (Honeybee) Apis cerana (Indian honeybee) Ara h 1AH11_ARAHY P43237 Allergen Ara h 1, Clone Arachis hypogaea P17 (Peanut)Ara h 1 AH12_ARAHY P43238 Allergen Ara h 1, Clone Arachis hypogaea P41b(Peanut) Ara t 8 PRO1_ARATH Q42449 Profilin 1 Arabidopsis thaliana(Mouse-ear cress) Asp f 1 RNMG_ASPRE P04389 Ribonuclease MitogillinAspergillus restrictus; [Precursor] Aspergillus fumigatus (Sartoryafumigata) Asp f 2 MAF2_ASPFU P79017 Major Allergen Asp f 2 Aspergillusfumigatus [Precursor] (Sartorya fumigata) Asp f 3 PM20_ASPFU O43099Probable Peroxisomal Aspergillus fumigatus Membrane Protein (Sartoryafumigata) PMP20 Asp f 13 AF13_ASPFU O60022 Allergen Asp f 13 Aspergillusfumigatus [Precursor] (Sartorya fumigata) Bet v 1 BV1A_BETVE P15494Major Pollen Allergen Betula verrucosa (White Bet v 1-a birch) (Betulapendula) Bet v 1 BV1C_BETVE P43176 Major Pollen Allergen Betulaverrucosa (White Bet v 1-c birch) (Betula pendula) Bet v 1 BV1D_BETVEP43177 Major Pollen Allergen Betula verrucosa (White Bet v 1-d/h birch)(Betula pendula) Bet v 1 BV1E_BETVE P43178 Major Pollen Allergen Betulaverrucosa (White Bet v 1-e birch) (Betula pendula) Bet v 1 BV1F_BETVEP43179 Major Pollen Allergen Betula verrucosa (White Bet v 1-f/i birch)(Betula pendula) Bet v 1 BV1G_BETVE P43180 Major Pollen Allergen Betulaverrucosa (White Bet v 1-g birch) (Betula pendula) Bet v 1 BV1J_BETVEP43183 Major Pollen Allergen Betula verrucosa (White Bet v 1-j birch)(Betula pendula) Bet v 1 BV1K_BETVE P43184 Major Pollen Allergen Betulaverrucosa (White Bet v 1-k birch) (Betula pendula) Bet v 1 BV1L_BETVEP43185 Major Pollen Allergen Betula verrucosa (White Bet v 1-1 birch)(Betula pendula) Bet v 1 BV1M_BETVE P43186 Major Pollen Allergen Betulaverrucosa (White Bet v 1-m/n birch) (Betula pendula) Bet v 2 PROF-BETVEP25816 Profilin Betula verrucosa (White birch) (Betula pendula) Bet v 3BTV3_BETVE P43187 Allergen Bet v 3 Betula verrucosa (White birch)(Betula pendula) Bla g 2 ASP2_BLAGE P54958 Aspartic Protease Bla gBlattella germanica 2 [Precursor] (German cockroach) Bla g 4 BLG4_BLAGEP54962 Allergen Bla g 4 Blattella germanica [Precursor] [Fragment](German cockroach) Bla g 5 GTS1_BLAGE O18598 Glutathione-S- Blattellagermanica transferase (German cockroach) Blo t 12 BT12_BLOTA Q17282Allergen Blo t 12 Blomia tropicalis (Mite) [Precursor] Bos d 2ALL2_BOVIN Q28133 Allergen Bos d 2 Bos taurus (Bovine) [Precursor] Bos d5 LACB_BOVIN P02754 Beta-lactoglobulin Bos taurus (Bovine) [Precursor]Bra j 1 ALL1_BRAJU P80207 Allergen Bra j 1-e, Brassica juncea (LeafSmall and Large Chains mustard) (Indian mustard) Can a 1 ADH1_CANALP43067 Alcohol Dehydrogenase Candida albicans 1 (Yeast) Can f 1ALL1_CANFA O18873 Major Allergen Can f 1 Canis famiiaris (Dog)[Precursor] Can f 2 ALL2_CANFA O18874 Minor Allergen Can f 2 Canisfamiliaris (Dog) [Precursor] Car b 1 MPA1_CARBE P38949 Major PollenAllergen Carpinus betulus Car b 1, Isoforms 1A (Hornbeam) and 1B Car b 1MPA2_CARBE P38950 Major Pollen Allergen Carpinus betulus Car b 1,Isoform 2 (Hornbeam) Cha o 1 MPA1_CHAOB Q96385 Major Pollen AllergenChamaecyparis obtusa Cha o 1 [Precursor] (Japanese cypress) Cla h 3DHAL_CLAHE P40108 Aldehyde Cladosporium herbarum Dehydrogenase Cla h 3RLA3_CLAHE P42038 60S Acidic Ribosomal Cladosporium herbarum Protein P2Cla h 4 HS70_CLAHE P40918 Heat Shock 70 KDa Cladosporium herbarumProtein Cla h 4 RLA4_CLAHE P42039 60S Acidic Ribosomal Cladosporiumherbarum Protein P2 Cla h 5 CLH5_CLAHE P42059 Minor Allergen Cla h 5Cladosporium herbarum Cla h 6 ENO_CLAHE P42040 Enolase Cladosporiumherbarum Cla h 12 RLA1_CLAHE P50344 60S Acidic Ribosomal Cladosporiumherbarum Protein P1 Cop c 2 THIO_CAPCM Cor a 1 MPAA_CORAV Q08407 MajorPollen Allergen Corylus avellana Cor a 1, Isoforms 5, 6, (Europeanhazel) 11 and 16 Cup a 1 MPA1_CUPAR Q9SCG9 Major Pollen AllergenCupressus arizonica Cup a 1 Cry j 1 SBP_CRYJA P18632 Sugi Basic ProteinCryptomeria japonica [Precursor] (Japanese cedar) Cry j 2 MPA2_CRYJAP43212 Possible Cryptomeria japonica Polygalacturonase (Japanese cedar)Cyn d 12 PROF_CYNDA O04725 Profilin Cynodon dactylon (Bermuda grass) Dacg 2 MPG2_DACGL Q41183 Pollen Allergen Dac g 2 Dactylis glomerata[Fragment] (Orchard grass) (Cocksfoot grass) Dau c 1 DAU1_DAUCA O04298Major Allergen Dau c 1 Daucus carota (Carrot) Der f 1 MMAL_DERFA P16311Major Mite Fecal Dermatophagoides Allergen Der f 1 farinae (House-dust[Precursor] mite) Der f 2 DEF2_DERFA Q00855 Mite Allergen Der f 2Dermatophagoides [Precursor] ferinae (House-dust mite) Der f 3DEF3_DERFA P49275 Mite Allergen Der f 3 Dermatophagoides [Precursor]ferinae (House-dust mite) Der f 6 DEF6_DERFA P49276 Mite Allergen Der f6 Dermatophagoides [Fragment] ferinae (House-dust mite) Der f 7DEF7_DERFA Q26456 Mite Allergen Der f 7 Dermatophagoides [Precursor]ferinae (House-dust mite) Der m 1 MMAL_DERMI P16312 Major Mite FecalDermatophagoides Allergen Der m 1 microceras (House-dust [Fragment]mite) Der p 1 MMAL_DERPT P08176 Major Mite Fecal DermatophagoidesAllergen Der p 1 pteronyssinus (House- [Precursor] dust mite) Der p 2DER2_DERPT P49278 Mite Allergen Der p 2 Dermatophagoides [Precursor]pteronyssinus (House- dust mite) Der p 3 DER3_DERPT P39675 Mite AllergenDer p 3 Dermatophagoides [Precursor] pteronyssinus (House- dust mite)Der p 4 AMY_DERPT P49274 Alpha-Amylase Dermatophagoides [Fragment]pteronyssinus (House- dust mite) Der p 5 DER5_DERPT P14004 Mite AllergenDer p 5 Dermatophagoides pteronyssinus (House- dust mite) Der p 6DER6_DERPT P49277 Mite Allergen Der p 6 Dermatophagoides [Fragment]pteronyssinus (House- dust mite) Der p 7 DER7_DERPT P49273 Mite AllergenDer p 7 Dermatophagoides [Precursor] pteronyssinus (House- dust mite)Dol a 5 VA5_DOLAR Q05108 Venom Allergen 5 Dolichovespula arenaria(Yellow hornet) Dol m 1 PA11_DOLMA Q06478 Phospholipase A1 1Dolichovespula [Precursor] [Fragment] maculata (White-face hornet)(Bald-faced hornet) Dol m 1 PA12_DOLMA P53357 Phospholipase A1 2Dolichovespula maculata (White-face hornet) (Bald-faced hornet) Dol m 2HUGA_DOLMA P49371 Hyaluronoglucosaminidase Dolichovespula maculata(White-face hornet) (Bald-faced hornet) Dol m 5 VA52_DOLMA P10736 VenomAllergen 5.01 Dolichovespula [Precursor] maculata (White-face hornet)(Bald-faced hornet) Dol m 5 VA53_DOLMA P10737 Venom Allergen 5.02Dolichovespula [Precursor] [Fragment] maculata (White-face hornet)(Bald-faced hornet) Equ c 1 ALL1_HORSE Q95182 Major Allergen Equ c 1Equus caballus (Horse) [Precursor] Equ c 2 AL21_HORSE P81216 Dandermajor Allergen Equus caballus (Horse) Equ c 2.0101 [Fragment] Equ c 2AL22_HORSE P81217 Dander Major Allergen Equus caballus (Horse) Equ c2.0102 [Fragment] Eur m 1 EUM1_EURMA P25780 Mite Group I AllergenEuroglyphus maynei Eur m 1 [Fragment] (House-dust mite) Fel d 1FELA_FELCA P30438 Major Allergen I Felis silvestris catus PolypeptideChain 1 (Cat) Major Form [Precursor] Fel d 1 FELB_FELCA P30439 MajorAllergen I Felis silvestris catus Polypeptide Chain 1 (Cat) Minor Form[Precursor] Fel d 1 FEL2_FELCA P30440 Major Allergen I Felis silvestriscatus Polypeptide Chain 2 (Cat) [Precursor] Gad c 1 PRVB_GADCA P02622Parvalbumin Beta Gadus callarias (Baltic cod) Gal d 1 IOVO_CHICK P01005Ovomucoid [Precursor] Gallus gallus (Chicken) Gal d 2 OVAL_CHICK P01012Ovalbumin Gallus gallus (Chicken) Gal d 3 TRFE_CHICK P02789Ovotransferrin Gallus gallus (Chicken) [Precursor] Gal d 4 LYC_CHICKP00698 Lysozyme C Gallus gallus (Chicken) [Precursor] Hel a 2 PROF_HELANO81982 Profilin Helianthus annuus (Common sunflower) Hev b 1 REF_HEVBRP15252 Rubber Elongation Hevea brasiliensis (Para Factor Protein rubbertree) Hev b 5 HEV5_HEVBR Q39967 Major Latex Allergen Hevea brasiliensis(Para Hev b 5 rubber tree) Hol l 1 MPH1_HOLLA P43216 Major PollenAllergen Holcul lanatus (Velvet Hol l 1 [Precursor] grass) Hor v 1IAA1_HORVU P16968 Alpha-amylase Inhibitor Hordeum vulgare Bmai-1[Precursor] (Barley) [Fragment] Jun a 1 MPA1_JUNAS P81294 Major PollenAllergen Juniperus ashei (Ozark Jun a 1 [Precursor] white cedar) Jun a 3PRR3_JUNAS P81295 Pathogenesis-Related Juniperus ashei (Ozark Protein[Precursor] white cedar) Lep d 1 LEP1_LEPDS P80384 Mite Allergen Lep d 1Lepidoglyphus [Precursor] destructor (Storage mite) Lol p 1 MPL1_LOLPRP14946 Pollen Allergen Lol p 1 Lolium perenne [Precursor] (Perennialryegrass) Lol p 2 MPL2_LOLPR P14947 Pollen Allergen Lol p 2- Loliumperenne a (Perennial ryegrass) Lol p 3 MPL3_LOLPR P14948 Pollen AllergenLol p 3 Lolium perenne (Perennial ryegrass) Lol p 5 MP5A_LOLPR Q40240Major Pollen Allergen Lolium perenne Lol p 5a [Precursor] (Perennialryegrass) Lol p 5 MP5B_LOLPR Q40237 Major Pollen Allergen Lolium perenneLol p 5b [Precursor] (Perennial ryegrass) Mal d 1 MAL1_MALDO P43211Major Allergen Mal d 1 Malus domestica (Apple) (Malus sylvestris) Mer a1 PROF_MERAN O49894 Profilin Mercurialis annua (Annual mercury) Met e 1TPM1_METEN Q25456 Tropomyosin Metapenaeus ensis (Greasyback shrimp)(Sand shrimp) Mus m 1 MUP6_MOUSE P02762 Major Urinary Protein 6 Musmusculus (Mouse) [Precursor] Myr p 1 MYR1_MYRPI Q07932 Major AllergenMyr p 1 Myrmecia pilosula [Precursor] (Bulldog ant) (Australian jumperant) Myr p 2 MYR2_MYRPI Q26464 Allergen Myr p 2 Myrmecia pilosula[Precursor] (Bulldog ant) (Australian jumper ant) Ole e 1 ALL1_OLEEUP19963 Major Pollen Allergen Olea europaea (Common olive) Ole e 4ALL4_OLEEU P80741 Major Pollen Allergen Olea europaea Ole e 4[Fragments] (Common olive) Ole e 5 SODC_OLEEU P80740 SuperoxideDismutase Olea europaea [CU-ZN] [Fragment] (Common olive) Ole e 7ALL7_OLEEU P81430 Pollen Allergen Ole e 7 Olea europaea [Fragment](Common olive) Ory s 1 MPO1_ORYSA Q40638 Major Pollen Allergen Oryzasativa (Rice) Ory s 1 [Precursor] Par j 1 NL11_PARJU P43217 ProbableNonspecific Parietaria judaica Lipid-Transfer Protein [Fragment] Par j 1NL12_PARJU O04404 Probable Nonspecific Parietaria judaica Lipid-TransferProtein 1 [Precursor] Par j 1 NL13_PARJU Q40905 Probable NonspecificParietaria judaica Lipid-Transfer Protein 1 [Precursor] Par j 2NL21_PARJU P55958 Probable Nonspecific Parietaria judaica Lipid-TransferProtein 2 [Precursor] Par j 2 NL22_PARJU O04403 Probable NonspecificParietaria judaica Lipid-Transfer Protein 2 [Precursor] Pha a 1MPA1_PHAAQ Q41260 Major Pollen Allergen Phalaris aquatica Pha a 1[Precursor] Pha a 5 MP51_PHAAQ P56164 Major Pollen Allergen Phalarisaquatica Pha a 5.1 [Precursor] Pha a 5 MP52_PHAAQ P56165 Major PollenAllergen Phalaris aquatica Pha a 5.2 [Precursor] Pha a 5 MP53_PHAAQP56166 Major Pollen Allergen Phalaris aquatica Pha a 5.3 [Precursor] Phaa 5 MP54_PHAAQ P56167 Major Pollen Allergen Phalaris aquatica Pha a 5.4[Fragment] Phl p 1 MPP1_PHLPR P43213 Pollen Allergen Phl p 1 Phleumpratense [Precursor] (Common timothy) Phl p 2 MPP2_PHLPR P43214 PollenAllergen Phl p 2 Phleum Pratense [Precursor] (Common timothy) Phl p 5MP5A_PHLPR Q40962 Pollen Allergen Phl p Phleum pratense 5a [Fragment](Common timothy) Phl p 5 MP5B_PHLPR Q40963 Pollen Allergen Phl p Phleumpratense 5b [Precursor] (Common timothy) [Fragment] Phl p 6 MPP6_PHLPRP43215 Pollen Allergen Phl p 6 Phleum pratense [Precursor] (Commontimothy) Phl p 11 PRO1_PHLPR P35079 Profilin 1 Phleum pratense (Commontimothy) Phl p 11 PRO2_PHLPR O24650 Profilin 2/4 Phleum pratense (Commontimothy) Phl p 11 PRO3_PHLPR O24282 Profilin 3 Phleum pratense (Commontimothy) Poa p 9 MP91_POAPR P22284 Pollen Allergen Kbg 31 Poa pratensis(Kentucky [Precursor] bluegrass) Poa p 9 MP92_POAPR P22285 PollenAllergen Kbg 41 Poa pratensis (Kentucky [Precursor] bluegrass) Poa p 9MP93_POAPR P22286 Pollen Allergen Kbg 60 Poa pratensis (Kentucky[Precursor] bluegrass) Pol a 5 VA5_POLAN Q05109 Venom Allergen 5Polistes annularis [Precursor] [Fragment] (Paper wasp) Pol d 5 VA5_POLDOP81656 Venom Allergen 5 Polistes dominulus (European paper wasp) Pol e 5VA5_POLEX P35759 Venom Allergen 5 Polistes exclamans (Paper wasp) Pol f5 VA5_POLFU P35780 Venom Allergen 5 Polistes fuscatus (Paper wasp) Pru a1 PRU1_PRUAV O24248 Major Allergen Pru a 1 Prunus avium (Cherry) Rat n 1MUP_RAT P02761 Major Urinary Protein Rattus norvegicus (Rat) [Precursor]Sol i 2 VA2_SOLIN P35775 Venom Allergen II Solenopsis invicta (Red[Precursor] imported fire ant) Sol i 3 VA3_SOLIN P35778 Venom AllergenIII Solenopsis invicta (Red imported fire ant) Sol i 4 VA4_SOLIN P35777Venom Allergen IV Solenopsis invicta (Red imported fire ant) Sol r 2VA2_SOLRI P35776 Venom Allergen II Solenopsis richteri (Black importedfire ant) Sol r 3 VA3_SOLRI P35779 Venom Allergen III Solenopsisrichteri (Black imported fire ant) Ves c 5 VA51_VESCR P35781 VenomAllergen 5.01 Vespa crabro (European hornet) Ves c 5 VA52_VESCR P35782Venom Allergen 5.02 Vespa crabro (European hornet) Ves f 5 VA5_VESFLP35783 Venom Allergen 5 Vespula flavopilosa (Yellow jacket) (Wasp) Ves g5 VA5_VESGE P35784 Venom Allergen 5 Vespula germanica (Yellow jacket)(Wasp) Ves m 1 PA1_VESMC P51528 Phospholipase A1 Vespula maculifrons(Eastern yellow jacket) (Wasp) Ves m 5 VA5_VESMC P35760 Venom Allergen 5Vespula maculifrons (Eastern yellow jacket) (Wasp) Ves p 5 VA5_VESPEP35785 Venom Allergen 5 Vespula pensylvanica (Western yellow jacket)(Wasp) Ves s 5 VA5_VESSQ P35786 Venom Allergen 5 Vespula squamosa(Southern yellow jacket) (Wasp) Ves v 1 PA1_VESVU P49369 PhospholipaseA1 Vespula vulgaris [Precursor] (Yellow jacket) (Wasp) Ves v 2HUGA_VESVU P49370 Hyaluronoglucosaminidase Vespula vulgaris (Yellowjacket) (Wasp) Ves v 5 VA5_VESVU Q05110 Venom Allergen 5 Vespulavulgaris [Precursor] (Yellow jacket) (Wasp) Ves vi 5 VA5_VESVI P35787Venom Allergen 5 Vespula vidua (Yellow jacket) (Wasp) Vesp m 5 VA5_VESMAP81657 Venom Allergen 5 Vespa mandarinia (Hornet) Zea m 1 MPZ1_MAIZEQ07154 Pollen Allergen Zea m Zea mays (Maize) 1

[0142] In other embodiments, the amino acid sequence of the secondpolypeptide of the fusion molecule is defined with reference to anautoantigen sequence. Examples of autoantigen sequences are listed inTable 2 below. Portions of the autoantigens listed in Table 2 are alsosuitable for use in the fusion polypeptides, wherein the portion retainsat least one autoantigen epitope, and retains the ability tospecifically bind the autoantibody or autoreactive T-cell receptor. Forexample, useful portions of the multiple sclerosis autoantigensmyelin-basic-protein (amino acids 83-99), proteolipid protein (aminoacids 139-151) and myelin oligodendrocyte glycoprotein (amino acids92-106) are known, where the portions retain at least one autoantigenicepitope. TABLE 2 Autoimmune Reference and/or GenBank AccessionAuto-antigen Disease(s) No. acetylcholine receptor (AChR) myastheniagravis Patrick and Lindstrom, Science 180:871-872 (1973); Lindstrom etal., Neurology 26:1054- 1059 (1976); Protti et al., Immunol. Today,15(1):41-42 (1994); Q04844; P02708; ACHUA1; AAD14247 gravin Nauert etal., Curr. Biol., 7(1):52-62 (1997); Q02952; AAB58938 titin (connectin)Gautel et al., Neurology 43:1581-1585 (1993); Yamamoto et al., Arch.Neurol., 58(6):869-870 (2001); AAB28119 neuronal voltage-gatedLambert-Eaton myasthenic Rosenfeld et al., Ann. Neurol., 33(1):113-120calcium channel syndrome (1993); A48895 CNS myelin-basic-proteinmultiple sclerosis Warren et al., Proc. Natl. Acad. Sci. USA (MBP),MBP₈₃₋₉₉ epitope 92:11061-11065 [1995]; Wucherpfennig et al., J. Clin.Invest., 100(5):1114-1122 [1997]; Critchfield et al., Science263:1139-1143 [1994]; Racke et al., Ann. Neurol., 39(1):46- 56 [1996];XP_040888; AAH08749; P02686 proteolipid protein (PLP), XP_010407PLP₁₃₉₋₁₅₁ epitope PLP₁₇₈₋₁₉₁ epitope myelin oligodendrocyte XP_041592glycoprotein (MOG), MOG₉₂₋₁₀₆ epitope αβ-crystallin Van Noort et al.,Nature 375:798 (1995); Van Sechel et al., J. Immunol., 162:129-135(1999); CYHUAB myelin-associated Latov, Ann. Neurol., 37(Suppl.1):S32-S42 glycoprotein (MAG), Po (1995); Griffin, Prog. Brain Res.,101:313- glycoprotein and PMP22 323 (1994); Rose and MacKay (Eds.), TheAutoimmune Diseases, Third Edition, Academic Press, p. 586-602 [1998];XP_012878; P20916 2′,3′-cyclic nucleotide 3′- P09543; JC1517phosphohydrolase (CNPase) glutamic acid decarboxylase type-I (insulindependent) Yoon et al., Science 284:1183-1187 [1999]; (GAD), and variousisoforms diabetes mellitus, also Stiff-Man Nepom et al., Proc. Natl.Acad. Sci. USA (e.g., 65 and 67 kDa isoforms) Syndrome (GAD) and other98(4):1763-1768 [2001]; Lernmark, J. Intern. diseases (GAD) Med.,240:259-277 [1996]; B41935; A41292; P18088; Q05329 insulin Wong et al.,Nature Med., 5:1026-1031 [1999]; Castaño et al., Diabetes 42:1202-1209(1993) 64 kD islet cell antigen/ Rabin et al., Diabetes 41:183-186(1992); tyrosine phosphatase-like islet Rabin et al., J. Immunol.,152:3183-3187 cell antigen-2 (IA-2, also (1994); Lan et al., DNA CellBiol., 13:505- termed ICA512) 514 (1994) phogrin (IA-2β) Wasmeier andHutton, J. Biol. Chem., 271:18161-18170 (1996); Q92932 type II collagenrheumatoid arthritis Cook et al., J. Rheumatol., 21:1186-1191 (1994);and Terato et al., Arthritis Rheumatol., 33:1493-1500 (1990) humancartilage gp39 P29965; XP_042961 (HCgp39) gp 130-RAPS P40189; BAA78112scl-70 antigen/topoisomerase-I scleroderma (systemic sclerosis), Douvaset al., J. Biol. Chem., 254:10514- various connective tissue 10522(1979); Shero et al., Science 231:737- diseases 740 (1986); P11387topoisomerase II (α/β) Meliconi et al., Clin. Exp. Immunol.,76(2):184-189(1989); XP_008649; NP_001059; Q02880 type I collagen Rienteet al., Clin. Exp. Immunol., 102(2):354-359 (1995); XP_037912fibrillarin, U3-small nuclear Arnett et al., Arthritis Rheum.,39:151-160 protein (snoRNP) (1996) Jo-1 antigen/aminoacyl polymyositis,dermatomyositis, Mathews and Bernstein, Nature 304:177-179 histidyl-tRNAsynthetase interstitial lung disease, (1983); Bernstein, Bailliere'sClin. Neurol., PL-7 antigen/threonyl tRNA Raynaud's phenomenon, also2:599-616 (1993); Targoff, J. Immunol., synthetase scleroderma (PM-scl)144(5):1737-1743 (1990); Targoff, J. Invest. PL-12 antigen/alanyl tRNADermatol., 100:116S-123S (1995); Rider and synthetase Miller, Clin.Diag. Lab. Immunol., 2:1-9 EJ antigen/glycyl-tRNA (1995); Targoff, J.Invest. Dermatol., synthetase 100:116S-123S (1995); von Muhlen and Tan,OJ antigen/NJ antigen Semin. Arthritis Rheum., 24:323-358 (1995);isoleucyl-tRNA synthetase Targoff et al., J. Clin. Invest., 84:162-172signal recognition particle (1989) (SRP) Mi-2 helicase PM-scl proteins(75 kDa, 100 kDa KJ antigen Fer antigen/ elongation fractor 1α Masantigen/ tRNA^(Ser) type IV collagen α3 chain Goodpasture syndromeHellmark et al., Kidney Int., 46:823-829 (1994); Q01955 Smith (Sm)antigens and systemic lupus erythematosus, Lerner and Steitz, Proc.Natl. Acad. Sci. USA snRNP's, including snRNPs mixed connective tissuedisease 76:5495-5499 (1979); Reuter et al., Eur. J. D1, D2, D3, B, B′,B3 (N), E, (MCTD), progressive systemic Immunol., 20:437-440 (1990);Petersson et F, and G, as found in RNP sclerosis, rheumatoid arthritis,al., J. Biol. Chem., 259:5907-5914 (1984) complexes U1, U2, U4/6, anddiscoid lupus erythematosus, U5. Sjögren's syndrome nRNP U1-snRNPcomplex, Klein et al., Clin. Exp. Rheumatol., 15:549- including subunitsU1-70 kD, 560 (1997) A and C. deoxyribonucleic acid (DNA), systemiclupus erythematosus Pisetsky, Curr. Top. Microl. Immunol.,double-stranded B-form 247:143-155 (2000); Radic et al., Crit. Rev.deoxyribonucleic acid (DNA), Immunol., 19(2):117-126 (1999)denatured/single-stranded Cyclin A autoimmune hepatic disease, andStrassburg et al., Gastroenterology 111:1582- other diseases 1592(1996); Strassburg et al., J. Hepatol., 25(6):859-866 (1996) Ro (SS-A)antigens Sjögren's syndrome, systemic Tan, Adv. Iminunol., 44:93-(1989);  52 kDa and cutaneous lupus McCauliffe and Sontheimer, J.Invest.  60 kDa erythematosis, rheumatoid Dermatol., 100:73S-79S (1993);Wolin and arthritis, neonatal lupus Steitz, Proc. Natl. Acad. Sci. USA81:1996- syndrome, polymyositis, 2000 (1984); Slobbe et al., Ann. Med.progressive systemic sclerosis, Interne., 142:592-600 (1991); AAB87094;primary biliary cirrhosis U01882; P10155 La (SS-B) antigen Sjögren'ssyndrome, neonatal Manoussakis et al., Scan. J. 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[0143] It is not intended that useful autoantigen sequences be limitedto those sequences provided in Table 2, as methods for theidentification of additional autoantigens are known in the art, e.g.,SEREX techniques (serological identification of antigens by recombinantexpression cloning), where expression libraries are screened usingautoimmune sera probes (Bachmann et al., Cell 60:85-93 [1990]; andPietromonaco et al., Proc. Natl. Acad. Sci. USA 87:1811-1815 [1990];Folgori et al., EMBO J, 13:2236-2243 [1994]). Similarly, it is notintended that the autoimmune diseases that can be treated using thecompositions and methods of the invention be limited to the diseaseslisted in Table 2, as additional diseases which have autoimmuneetiologies will be identified in the future.

[0144] In some embodiments of the invention, the first polypeptidesequence present in the fusion molecule may comprise a sequence encodedby a nucleic acid hybridizing under stringent conditions to thecomplement of the coding sequence of a native γhinge-CHγ2-CHγ3 sequence,preferably the γhinge-CHγ2-CHγ3 coding sequence from within SEQ ID NO:1, or with the coding sequence of another immunoglobulin heavy chainconstant region sequence required for IgG binding. When the firstpolypeptide sequence binds specifically to an ITIM-containing receptorexpressed on mast cells, basophils or B cells, it is preferably encodedby nucleic acid hybridizing under stringent conditions to the complementof the coding sequence of a native ligand of that receptor.

[0145] Similarly, the second polypeptide sequence present in the fusionmolecules of the invention may comprise a sequence encoded by nucleicacid hybridizing under stringent conditions to the complement of thecoding sequence of a native CHε2-CHε3-CHε4 sequence, preferably theCHε2-CHε3-CHε4 coding sequence from within SEQ ID NO: 4, or to thecomplement of the coding sequence of a native allergen or autoantigen,such as those listed in Tables 1 and 2.

[0146] Whenever the first and/or second polypeptide sequence included inthe fusion molecules of the invention is an amino acid variant of anative immunoglobulin constant region sequence, it is required to retainthe ability to bind to the corresponding native receptor, such as anative IgG inhibitory receptor (e.g. FcγRIIb) and a native high-affinityIgE receptor (e.g. FcεR1) or native low-affinity IgE receptor (FcεR11,CD23), respectively. As discussed above, the receptor binding domainswithin the native IgG and IgE heavy chain constant region sequences havebeen identified. Based on this knowledge, the amino acid sequencevariants may be designed to retain the native amino acid residuesessential for receptor binding, or to perform only conservative aminoacid alterations (e.g. substitutions) at such residues.

[0147] In making amino acid sequence variants that retain the requiredbinding properties of the corresponding native sequences, thehydropathic index of amino acids may be considered. For example, it isknown that certain amino acids may be substituted for other amino acidshaving a similar hydropathic index or score without significant changein biological activity. Thus, isoleucine, which has a hydrophatic indexof +4.5, can generally be substituted for valine (+4.2) or leucine(+3.8), without significant impact on the biological activity of thepolypeptide in which the substitution is made. Similarly, usually lysine(−3.9) can be substituted for arginine (−4.5), without the expectationof any significant change in the biological properties of the underlyingpolypeptide. Other considerations for choosing amino acid substitutionsinclude the similarity of the side-chain substituents, for example,size, electrophilic character, charge in various amino acids. Ingeneral, alanine, glycine and serine; arginine and lysine; glutamate andaspartate; serine and threonine; and valine, leucine and isoleucine areinterchangeable, without the expectation of any significant change inbiological properties. Such substitutions are generally referred to asconservative amino acid substitutions, and, as noted above, are thepreferred type of substitutions within the polypeptides of the presentinvention.

[0148] Alternatively or in addition, the amino acid alterations mayserve to enhance the receptor binding properties of the fusion moleculesof the invention. Variants with improved receptor binding and, as aresult, superior biological properties can be readily designed usingstandard mutagenesis techniques, such as alanine-scanning mutagenesis,PCR mutagenesis or other mutagenesis techniques, coupled with receptorbinding assays, such as the assay discussed below or described in theExample.

[0149] In a preferred embodiment, the fusion molecules of the presentinvention comprise a first polypeptide sequence including functionallyactive hinge, CH2 and CH3 domains of the constant region of an IgG₁heavy chain (γhinge-CHγ2-CHγ3 sequence) linked at its C-terminus to theN-terminus of a second polypeptide including functionally active CH2,CH3 and CH4 domains of the constant region of an IgE heavy chain(CHε2-CHε3-CHε4 sequence). In a particularly preferred embodiment, thefirst polypeptide sequence is composed of functionally active hinge, CH2and CH3 regions of a native human IgG₁ heavy chain, linked at itsC-terminus to the N-terminus of a second polypeptide composed offunctionally active CH2, CH3 and CH4 domains of a native human IgE heavychain constant region.

[0150] While it is preferred to fuse the IgG heavy chain constant regionsequence (or a homologous sequence) C-terminally to the N-terminus ofthe IgE heavy chain constant region sequence (or a homologous sequence),fusion molecules in which the IgE heavy chain constant region sequence(or a homologous sequence) is fused C-terminally to the N-terminus ofthe IgG heavy chain constant region sequence (or a homologous sequence)are also within the scope of the invention. The fusion molecules mayalso comprise repeats of identical or different IgG and/or IgE heavychain constant region sequences. For example, two repeats of IgG heavychain constant region sequences, each including an IgG inhibitoryreceptor-binding domain, can be followed by IgE heavy chain constantregion sequences (GGE structure), or two repeats of identical ordifferent IgG heavy chain constant region sequences may flank an IgEheavy chain constant region sequence (GEG structure), etc.

[0151] Fusion molecules comprising more than one binding sequence for atarget receptor (e.g. an FcγRIIb receptor) are expected to have superiorbiological, e.g. anti-allergic properties.

[0152] The same considerations apply to the structure of fusionmolecules where the second polypeptide sequence comprises, is or isderived from an allergen or autoantigen protein. Such molecules may alsoinclude repeats of the IgG heavy chain constant region sequences, fusedto either or both sides of the allergen sequence.

[0153] Similarly, molecules in which the first polypeptide sequencebinds to a different inhibitory receptor expressed on mast cells and/orbasophils, e.g. an ITIM-containing inhibitory receptor functionallyconnected to a second polypeptide sequence binding directly orindirectly to an IgE receptor, e.g. FcεR1, may contain multiple repeatsof the inhibitory receptor binding regions and/or the IgE bindingregions.

[0154] In all embodiments, the two polypeptide sequences arefunctionally connected, which means that they retain the ability to bindto the respective native receptors, such as a native IgG inhibitoryreceptor, e.g. a low-affinity FcγRIIb receptor, and to a nativehigh-affinity IgE receptor, e.g. FcεRI or low-affinity IgE receptor, asdesired. As a result, the fusion molecules, comprising the first andsecond polypeptide sequences functionally connected to each other, arecapable of cross-linking the respective native receptors, such asFcγRIIb and FcεRI or FcγRIIb and FcεRII. In order to achieve afunctional connection between the two binding sequences within thefusion molecules of the invention, it is preferred that they retain theability to bind to the corresponding receptor with a binding affinitysimilar to that of a native immunoglobulin ligand of that receptor.

[0155] The fusion molecules of the present invention are typicallyproduced and act as homodimers or heterodimers, comprising two of thefusion molecules hereinabove described covalently linked to each other.The covalent attachment is preferably achieved via one or more disulfidebonds. For example, the prototype protein designated GE2 is produced asa homodimer composed of the two γhinge-CHγ2-CHγ3-15aalinker-CHε2-CHε3-CHε4 chains connected to each other by interchaindisulfide bonds, to provide an immunoglobulin-like structure. It is alsopossible to produce heterodimers, in which two different fusionmolecules are linked to each other by one or more covalent linkages,e.g. disulfide bond(s). Such bifunctional structures might beadvantageous in that they are able to cross-link the same or differentIgεR(s) with different inhibitory receptors.

[0156] Receptor binding can be tested using any known assay method, suchas competitive binding assays, direct and indirect sandwich assays.Thus, binding of a first polypeptide sequence included in the fusionmolecules herein to a low-affinity IgG inhibitory receptor, or thebinding of a second polypeptide sequence included herein to ahigh-affinity or low-affinity IgE receptor can be tested usingconventional binding assays, such as competitive binding assays,including RIAs and ELISAs. Ligand/receptor complexes can be identifiedusing traditional separation methods as filtration, centrifugation, flowcytometry, and the results from the binding assays can be analyzed usingany conventional graphical representation of the binding data, such asScatchard analysis. The assays may be performed, for example, using apurified receptor, or intact cells expressing the receptor. One or bothof the binding partners may be immobilized and/or labeled. A particularcell-based binding assay is described in the Example below.

[0157] The two polypeptide sequences present in the fusion molecules ofthe invention may be associated with one another by any means thatallows them to cross-link the relevant receptors. Thus, association maytake place by a direct or indirect covalent linkage, where “indirect”covalent linkage means that the two polypeptide sequences are part ofseparate molecules that interact with one another, either directly orindirectly. For example, each polypeptide sequence can be directlylinked to one member of an interacting pair of molecules, such as, forexample, a biotin/avidin pair.

[0158] Alternatively, the two polypeptide sequences can be linked usinga “dimerizer” system based on linkage to an entity that associates witha common ligand, such as dimerizer systems based on cyclosporine A,FK506, rapamycin, countermycin, and the like.

[0159] In a preferred embodiment, the first and second polypeptidesequences, such as, for example, two immunoglobulin constant regionsegments, or an immunoglobulin constant region sequence and an allergenor autoantibody sequence, are connected by a polypeptide linker. Thepolypeptide linker functions as a “spacer” whose function is to separatethe functional receptor binding domains, or the Fcγ receptor bindingdomain and the IgE-binding sequence in the allergen or autoantigen, sothat they can independently assume their proper tertiary conformation.The polypeptide linker usually comprises between about 5 and about 25residues, and preferably contains at least about 10, more preferably atleast about 15 amino acids, and is composed of amino acid residues whichtogether provide a hydrophilic, relatively unstructured region. Linkingamino acid sequences with little or no secondary structure work well.The specific amino acids in the spacer can vary, however, cysteinesshould be avoided. Suitable polypeptide linkers are, for example,disclosed in WO 88/09344 (published on Dec. 1, 1988), as are methods forthe production of multifunctional proteins comprising such linkers.

[0160] In one embodiment, the fusion molecule containing allergen orautoantigen sequence is designed to have a dual purpose, where thefusion molecule (a) attenuates the allergic response by cross-linkinginhibitory ITIM-containing receptors and stimulatory IgE receptors, aswell as (b) provides antigenic material suitable for use in traditionaldesensitisation immunotherapies. This dual function is of value, as itprovides material suitable for use in desensitisation therapy forallergic or autoimmune disease, and simultaneously has the inherentability to suppress possible anaphylactic reactions caused by theadministration of the antigen-containing fusion polypeptide to a subjectduring desensitisation immunotherapy. Desensitisation therapies,including those using the fusion polypeptide of the present invention,utilize a mechanism of polypeptide internalization, followed byintracellular processing and presentation on the surface of a cell(e.g., but not limited to, antigen presenting cells; APCs) in thecontext of class I or class II major histocompatibility complex (MHC Ior MHC II) molecules. It is the copresentation of antigen and MHC toT-cells that, under certain conditions known in the art, produces thedesirable effect of “tolerance” to that antigen.

[0161] When used as vaccine material for desensitisation therapy, thefusion polypeptide of the present invention invention is internalizedfollowing administration to a subject, and thus, becomes intracellular.The internalization can be by any mechanism, although mechanismscomprising endocytosis, phagocytosis, pinocytosis, or any othermechanism of receptor or non-receptor-mediated internalization arecontemplated. The internalization and subsequent processing of thefusion polypeptide is a requirement for presentation to T-cells.

[0162] Cell surface presentation of antigen by MHC I and MHC II utilizetwo distinct mechanisms.

[0163] MHC I presentation processes antigen from the endoplasmicreticulum and cytosol in an ATP-dependent manner. Briefly, this processentails the marking of antigens for degradation by ubiquitination,followed by proteolytic processing in a proteasome-dependent manner.Additional “trimming” proteases are also implicated in the generation ofpeptides suitable for copresentation with MHC I (Rock and Goldberg,Annu. Rev. Immunol., 17:739-779 [1999]; Pamer and Cresswell, Annu. Rev.Immunol., 16:323-358 [1998]; and Luckey et al., Jour. Immunol.,167:1212-1221 [2001]). In contrast, processing of antigens forcopresentation with MHC II utilizes endocytosis and anendosomal/lysosomal pathway that partitions antigens from the cytosol,and utilizes a number of distinct ATP-independent, acid-optimalproteases with various cleavage specificities (Watts, Annu. Rev.Immunol., 15:821-850 [1997]; and Watts, Curr. Opin. Immunol.,13:(1):26-31 [2001]).

[0164] Some of the signal sequences that mark MHC I antigens forprocessing via the proteasome pathway are known. It is recognized thatantigens with large, bulky or charged amino termini are ¢ rapidlyubiquitinated and degraded, whereas the same proteins with N-terminalmethionines or other small N-terminal residues are more resistant toubiquitin-mediated degradation (Varshavsky, Cell 69:725-735 [1992]).Furthermore, the proteasome has been shown to contain at least threedistinct protease activities. These are (1) a preference for peptidebonds following large hydrophobic residues (i.e., a chymotrypsin-likeactivity), (2) a cleavage specificity following basic residues, and (3)a cleavage preference following acidic residues (Rock and Goldberg,Annu. Rev. Immunol., 17:739-779 [1999]; Pamer and Cresswell, Annu. Rev.Immunol., 16:323-358 [1998]). It has been reported that these activitiesare allosterically controlled, and the chymotrypsin-like activityappears to be controlling or rate-limiting (Kisselev et al., Mol. Cell4(3):395-402 [1999]).

[0165] Intracellular proteases involved in the processing of antigenwithin specialized endosomal compartments for copresentation inconjunction with MHC II on APCs are also known, and their cleavagespecificities have been determined (Watts, Annu. Rev. Immunol.,15:821-850 [1997]; Villadangos et al., Immunol. Rev., 172:109-120[1999]; Antoniou et al, Immunity 12(4):391-398, [2000]; Villadangos andPloegh, Immunity 12(3):233-239 [2000]; and Watts, Curr. Opin. Immunol.,13:(1):26-31 [2001]). Many of these proteases involved in antigenprocessing in the endosomal degradation pathway are cysteine, aspartateor arginine endoproteases. Proteases involved in antigen processinginclude, but are not limited to, those listed in Table 3, below. TABLE 3Protease Recognition Motif Cathepsins B, C, F, H, K, L, L2, O cysteineproteases S, V and Z Cathepsin D aspartate proteases Cathepsin Easpartate protease legumain/hemoglobinase cysteine protease family/asparaginyl endopeptidase (AEP) asparagine residues Napsin A aspartateprotease Napsin B aspartate protease

[0166] It is contemplated that in some embodiments of this invention,the fusion polypeptide contains amino acid sequences that facilitateeither (a) protease cleavage of the linker, or (b) general proteolyticprocessing of the antigen, and thereby provides antigenic material thatis more readily processed and presented on the cell surface (e.g., onthe surface of an APC). In some embodiments, these proteolytic signalsare within the linker sequence joining the antigen and Fcγ portions ofthe fusion polypeptide. In other embodiments, the proteolysis-promotingsequences are located in other parts of the fusion polypeptide, forexample, in the N- or C-termini of the fusion polypeptide.

[0167] More specifically, it is contemplated that fusion polypeptides ofthe present invention can contain various amino acid sequences thatpromote ubiquitin-targetting of the polypeptide, and also can containvarious amino acid residues to target the polypeptide for proteasomeprocessing and MHC I copresentation. For example, the fusion polypeptidecan be constructed to contain large, bulky or charged amino acidresidues in the amino-terminus to promote ubiquitin targetting.Alternatively or concurrently, the fusion polypeptide can contain largehydrophobic, basic or acidic residues to direct proteasome cleavageanywhere in the fusion polypeptide, and most advantageously, within thepolypeptide linker region. However, it is not necessary to have anunderstanding of the molecular mechanisms of antigen processing andpresentation to make and use the present invention.

[0168] Similarly, it is contemplated that the fusion polypeptides of thepresent invention can contain various amino acid sequences for thepurpose of promoting endosomal/lysosomal proteolytic processing and MHCII copresentation. For example, the fusion polypeptide can be enrichedin cysteine, aspartate or arginine residues. In preferred embodiments,the linker region of the fusion polypeptide is enriched in theseresidues to facilitate cleavage of the fusion polypeptide into twohalves, where the half containing the allergen or autoantigen sequencecan be further processed and displayed on the APC in association withMHC II. However, it is not necessary to have an understanding of themolecular mechanisms of antigen processing and presentation to make anduse the present invention.

[0169] In a less preferred embodiment, the IgG and IgE constant regionsequences, the IgG constant region sequences and the allergen orautoantigen sequences, or sequences showing high degree of -sequenceidentity with such sequences, may be directly fused to each other, orconnected by non-polypeptide linkers. Such linkers may, for example, beresidues of covalent bifunctional cross-linking agents capable oflinking the two sequences without the impairment of the receptor(antibody) binding function. The bifunctional cross-linking reagents canbe divided according to the specificity of their functional groups, e.g.amino, sulfhydryl, guanidino, indole, carboxyl specific groups. Ofthese, reagents directed to free amino groups have become especiallypopular because of their commercial availability, ease of synthesis andthe mild reaction conditions under which they can be applied. A majorityof heterobifunctional cross-linking reagents contains a primaryamine-reactive group and a thiol-reactive group (for review, see Ji, T.H. “Bifunctional Reagents” in: Meth. Enzymol. 91:580-609 (1983)).

[0170] In a further specific embodiment, the two polypeptide sequences(including variants of the native sequences) are dimerized byamphiphilic helices. It is known that recurring copies of the amino acidleucine (Leu) in gene regulatory proteins can serve as teeth that “zip”two protein molecules together to provide a dimer. For further detailsabout leucine zippers, which can serve as linkers for the purpose of thepresent invention, see for example: Landschulz, W. H., et al. Science240:1759-1764 (1988); O'Shea, E. K. et al., Science 243: 38-542 (1989);McKnight, S. L., Scientific American 54-64, April 1991; Schmidt-Dorr. T.et al., Biochemistry 30:9657-9664 (1991); Blondel, A. and Bedouelle, H.Protein Engineering 4:457-461 (1991), and the references cited in thesepapers.

[0171] In a different approach, the two polypeptide sequences (includingvariants of the native sequences) are linked via carbohydate-directedbifunctional cross-linking agents, such as those disclosed in U.S. Pat.No. 5,329,028.

[0172] The cross-linking of an inhibitory receptor expressed on mastcells and/or basophils, such as an ITIM-containing receptor, includingIgG inhibitory receptors, e.g. FcγRIIb and a high-affinity IgE receptor,e.g. FcεRI or low-affinity IgE receptor, e.g. FcεRII, inhibit FcεRmediated biological responses. Such biological responses preferably arethe mediation of an allergic reactions or autoimmune reactions via FcεR,including, without limitation, conditions associated with IgE mediatedreactions, such as, for example, asthma, allergic rhinitis, foodallergies, chronic urticaria and angioedema, allergic reactions tohymenophthera (e.g. bee and yellow jacket) stings or medications such aspenicillin. These responses also include the severe physiologicalreaction of anaphylactic shock, which may occur upon inadvertentexposure to allergen (e.g., bee venom), or alternatively, may occur uponintentional administration of allergen or autoantigen, as during peptidetherapy for treatment of allergic conditions or autoimmune disease.

[0173] 2. Preparation of the Fusion Molecules

[0174] When the fusion molecules are polypeptides, in which the firstand second polypeptide sequences are directly fused or functionallyconnected by a polypeptide linker, they can be prepared by well knownmethods of recombinant DNA technology or traditional chemical synthesis.If the polypeptides are produced by recombinant host cells, cDNAencoding the desired polypeptide of the present invention is insertedinto a replicable vector for cloning and expression. As discussedbefore, the nucleotide and amino acid sequences of native immunoglobulinconstant regions, including native IgG and IgE constant regionsequences, are well known in the art and are readily available, forexample, from Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institute of Health,Bethesda, Md. (1991).

[0175] The sequences of a large number of allergens are also well knownin the art. According to a nomenclature system established for allergensby the WHO/IUIS Allergen Nomenclature Subcommittee, the designation ofany particular allergen is composed of the first three letters of thegenus; a space; the first letter of the species name; a space and anarabic number. In the event that two species names have identicaldesignations, they are discriminated from one another by adding one ormore letters to each species designation. Using this designation, theallergen Aln G 1 is a major pollen allergen from the genus Alnus and thespecies glutinosa, the sequence of which is available from theSWISS-PROT database under the entry name MPAC_ALNGL (Primary Accessionnumber: P38948) (Breitender et al., J. Allergy Clin. Immunol. 90:909-917(1992)). A list of known antigens, including their origin, entry nameand Primary Accession Number in the SWISS-PROT database is provided inTable 1. The molecular weight of most food allergens is between 10,000and 70,000 Da. Some allergens, such as Ara h 1 (63.5 kDa) and Ara h 2(17 kDa), occur as polymers that are larger, e.g. 200 to 300 kDa.

[0176] Similarly, a list of known autoantigens implicated in humandisease is provided in Table 2. This table lists the autoantigenname(s), and the disease states associated with the presence ofautoantibodies to the particular autoantigen. This table lists onlythose autoimmune diseases for which the molecular identification of theautoantigen has been made. As can be seen in the table, the assignmentof one particular autoantibody to one specific disease is frequentlycomplex, as patients with a single autoimmune disorder often show morethan one autoreactive antibody, and vice versa, a particular autoantigenmay be involved on more than one autoimmune disease. It is not intendedthat the invention be limited to the use of only those sequencesprovided in Table 2. As autoantigens are identified in additionalautoimmune diseases, those molecular sequences will also find use withthe invention.

[0177] As noted earlier, it might be advantageous to use in the fusionmolecules of the present invention a fragment of a native or variantallergen or autoantigen that contains only a single IgE-binding site orimmunodominant epitope. For many of the allergen proteins listed inTables 1 and 2, the IgE-binding sites and immunodominant epitopes havebeen determined. For example, the IgE-binding epitopes of Par j 2, amajor allergen of Parietaria judaica pollen, have been determined byCosta et al., Allergy 55:246-50 (2000). The IgE-binding epitopes ofmajor peanut antigens Ara h 1 (Burks et al., Eur. J. Biochem. 254:334-9(1997)); Ara h 2 (Stanley et al., Arch Biochem. Biophys. 342:244-53(1997)); and Ara h 3 (Rabjohn et al., J. Clin. Invest. 103:535-42(1999)) are also known, just to mention a few. Also, for the CNS myelinbasic protein (MBP) autoantigen, the immunodominant epitope has beenmapped to a small domain encompassing approximately amino acid positions83 through 99 (Ota et al, Nature 346:183-187 [1990]; Warren and Catz, J.Neuroimmunol., 39:81-90 [1992]; Warren and Catz, J. Neuroimmunol.,43:87-96 [1993]; and Warren et al., Proc. Natl. Acad. Sci. USA92:11061-11065 [1995]). Short synthetic peptides corresponding to thisepitope have been used in peptide immunotherapy for multiple sclerosis(e.g., Warren et al., J. Neurol. Sci., 152:31-38 [1997]).

[0178] Suitable vectors are prepared using standard techniques ofrecombinant DNA technology, and are, for example, described in“Molecular Cloning: A Laboratory Manual”, 2^(nd) edition (Sambrook etal., 1989); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “AnimalCell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology”(Academic Press, Inc.); “Handbook of Experimental Immunology”, 4 hedition (D. M. Weir & C. C. Blackwell, eds., Blackwell Science Inc.,1987); “Gene Transfer Vectors for Mammalian Cells” (J. M. Miller & M. P.Calos, eds., 1987); “Current Protocols in Molecular Biology” (F. M.Ausubel et al., eds., 1987); “PCR: The Polymerase Chain Reaction”,(Mullis et al., eds., 1994); and “Current Protocols in Immunology” (J.E. Coligan et al., eds., 1991). Isolated plasmids and DNA fragments arecleaved, tailored, and ligated together in a specific order to generatethe desired vectors. After ligation, the vector containing the gene tobe expressed is transformed into a suitable host cell.

[0179] Host cells can be any eukaryotic or prokaryotic hosts known forexpression of heterologous proteins. Accordingly, the polypeptides ofthe present invention can be expressed in eukaryotic hosts, such aseukaryotic microbes (yeast) or cells isolated from multicellularorganisms (mammalian cell cultures), plants and insect cells. Examplesof mammalian cell lines suitable for the expression of heterologouspolypeptides include monkey kidney CV1 cell line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney cell line 293S (Graham etal, J. Gen. Virol. 36:59 [1977]); baby hamster kidney cells (BHK, ATCCCCL 10); Chinese hamster ovary (CHO) cells (Urlaub and Chasin, Proc.Natl. Acad. Sci. USA 77:4216 [1980]; monkey kidney cells (CVI-76, ATCCCCL 70); African green monkey cells (VERO-76, ATCC CRL-1587); humancervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK,ATCC CCL 34); human lung cells (W138, ATCC CCL 75); and human livercells (Hep G2, HB 8065). In general myeloma cells, in particular thosenot producing any endogenous antibody, e.g. the non-immunoglobulinproducing myeloma cell line SP2/0, are preferred for the production ofthe fusion molecules herein.

[0180] Eukaryotic expression systems employing insect cell hosts mayrely on either plasmid or baculoviral expression systems. The typicalinsect host cells are derived from the fall army worm (Spodopterafrugiperda). For expression of a foreign protein these cells areinfected with a recombinant form of the baculovirus Autographacalifornica nuclear polyhedrosis virus which has the gene of interestexpressed under the control of the viral polyhedrin promoter. Otherinsects infected by this virus include a cell line known commercially as“High 5” (Invitrogen) which is derived from the cabbage looper(Trichoplusia ni). Another baculovirus sometimes used is the Bombyx morinuclear polyhedorsis virus which infect the silk worm (Bombyx mori).Numerous baculovirus expression systems are commercially available, forexample, from Invitrogen (Bac-N-Blue™), Clontech (BacPAK™ BaculovirusExpression System), Life Technologies (BAC-TO-BAC™), Novagen (Bac VectorSystem™), Pharmingen and Quantum Biotechnologies). Another insect cellhost is common fruit fly, Drosophila melanogaster, for which a transientor stable plasmid based transfection kit is offered commercially byInvitrogen (The DES™ System).

[0181]Saccharomyces cerevisiae is the most commonly used among lowereukaryotic hosts. However, a number of other genera, species, andstrains are also available and useful herein, such as Pichia pastoris(EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:165-278[1988]). Yeast expression systems are commercially available, and can bepurchased, for example, from Invitrogen (San Diego, Calif.). Otheryeasts suitable for bi-functional protein expression include, withoutlimitation, Kluyveromyces hosts (U.S. Pat. No. 4,943,529), e.g.Kluyveromyces lactis; Schizosaccharomyces pombe (Beach and Nurse, Nature290:140 (1981); Aspergillus hosts, e.g., A. niger (Kelly and Hynes, EMBOJ., 4:475-479 [1985]) and A. nidulans (Ballance et al., Biochem.Biophys. Res. Commun., 112:284-289 [1983]), and Hansenula hosts, e.g.,Hansenula polymorpha. Yeasts rapidly growth on inexpensive (minimal)media, the recombinant can be easily selected by complementation,expressed proteins can be specifically engineered for cytoplasmiclocalization or for extracellular export, and are well suited forlarge-scale fermentation.

[0182] Prokaryotes are the preferred hosts for the initial cloningsteps, and are particularly useful for rapid production of large amountsof DNA, for production of single-stranded DNA templates used forsite-directed mutagenesis, for screening many mutants simultaneously,and for DNA sequencing of the mutants generated. E. coli strainssuitable for the production of the peptides of the present inventioninclude, for example, BL21 carrying an inducible T7 RNA polymerase gene(Studier et al., Methods Enzymol., 185:60-98 [1990]); AD494 (DE3);EB105; and CB (E. coli B) and their derivatives; K12 strain 214 (ATCC31,446); W3110 (ATCC 27,325); X1776 (ATCC 31,537); HB11 (ATCC 33,694);JM101 (ATCC 33,876); NM522 (ATCC 47,000); NM538 (ATCC 35,638); NM539(ATCC 35,639), etc. Many other species and genera of prokaryotes may beused as well. Indeed, the peptides of the present invention can bereadily produced in large amounts by utilizing recombinant proteinexpression in bacteria, where the peptide is fused to a cleavable ligandused for affinity purification.

[0183] Suitable promoters, vectors and other components for expressionin various host cells are well known in the art and are disclosed, forexample, in the textbooks listed above.

[0184] Whether a particular cell or cell line is suitable for theproduction of the polypeptides herein in a functionally active form, canbe determined by empirical analysis. For example, an expressionconstruct comprising the coding sequence of the desired molecule may beused to transfect a candidate cell line. The transfected cells are thengrowth in culture, the medium collected, and assayed for the presence ofsecreted polypeptide. The product can then be quantitated by methodsknown in the art, such as by ELISA with an antibody specifically bindingthe IgG, IgE, or allergen portion of the molecule.

[0185] In certain instances, particularly when two polypeptide sequencesmaking up the bifunctional molecule of the present invention areconnected with a non-polypeptide linker, it may be advantageous toindividually synthesize the first and second polypeptide sequences, e.g.by any of the recombinant approaches discussed above, followed byfunctionally linking the two sequences.

[0186] Alternatively, the two polypeptide sequences, or the entiremolecule, may be prepared by chemical synthesis, such as solid phasepeptide synthesis. Such methods are well known to those skilled in theart. In general, these methods employ either solid or solution phasesynthesis =methods, described in basic textbooks, such as, for example,J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd Ed.,Pierce Chemical Co., Rockford, Ill. (1984) and G. Barany and R. B.Merrifield, The Peptide: Analysis Synthesis, Biology, editors E. Grossand J. Meienhofer, Vol. 2, Academic Press, New York, (1980), pp. 3-254,for solid phase peptide synthesis techniques; and M. Bodansky,Principles of Peptide Synthesis, Springer-Verlag, Berlin (1984) and E.Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis,Biology, supra, Vol. 1, for classical solution synthesis.

[0187] The fusion molecules of the present invention may include aminoacid sequence variants of native immunoglobulin (e.g., IgG and/or IgE),allergen (e.g., Ara h 2 sequences) or autoantigen (e.g., myelin basicprotein). Such amino acid sequence variants can be produced byexpressing the underlying DNA sequence in a suitable recombinant hostcell, or by in vitro synthesis of the desired polypeptide, as discussedabove. The nucleic acid sequence encoding a polypeptide variant ispreferably prepared by site-directed mutagenesis of the nucleic acidsequence encoding the corresponding native (e.g. human) polypeptide.Particularly preferred is site-directed mutagenesis using polymerasechain reaction (PCR) amplification (see, for example, U.S. Pat. No.4,683,195 issued Jul. 28, 1987; and Current Protocols In MolecularBiology, Chapter 15 (Ausubel et al., ed., 1991). Other site-directedmutagenesis techniques are also well known in the art and are described,for example, in the following publications: Current Protocols InMolecular Biology, supra, Chapter 8; Molecular Cloning: A LaboratoryManual., 2^(nd) edition (Sambrook et al., 1989); Zoller et al., MethodsEnzymol. 100:468-500 (1983); Zoller & Smith, DNA 3:479-488 (1984);Zoller et al., Nucl. Acids Res., 10:6487 (1987); Brake et al., Proc.Natl. Acad. Sci. USA 81:4642-4646 (1984); Botstein et al., Science229:1193 (1985); Kunkel et al., Methods Enzymol. 154:367-82 (1987),Adelman et al., DNA 2:183 (1983); and Carter et al., Nucl. Acids Res.,13:4331 (1986). Cassette mutagenesis (Wells et al., Gene, 34:315[1985]), and restriction selection mutagenesis (Wells et al., Philos.Trans. R. Soc. London SerA, 317:415 [1986]) may also be used.

[0188] Amino acid sequence variants with more than one amino acidsubstitution may be generated in one of several ways. If the amino acidsare located close together in the polypeptide chain, they may be mutatedsimultaneously, using one oligonucleotide that codes for all of thedesired amino acid substitutions. If, however, the amino acids arelocated some distance from one another (e.g., separated by more than tenamino acids), it is more difficult to generate a single oligonucleotidethat encodes all of the desired changes. Instead, one of two alternativemethods may be employed. In the first method, a separate oligonucleotideis generated for each amino acid to be substituted. The oligonucleotidesare then annealed to the single-stranded template DNA simultaneously,and the second strand of DNA that is synthesized from the template willencode all of the desired amino acid substitutions. The alternativemethod involves two or more rounds of mutagenesis to produce the desiredmutant.

[0189] The polypeptides of the invention can also be prepared by thecombinatorial peptide library method disclosed, for example, inInternational Patent Publication PCT WO 92/09300. This method isparticularly suitable for preparing and analyzing a plurality ofmolecules, that are variants of a given predetermined sequences, and is,therefore, particularly useful in identifying polypeptides with improvedbiological properties, which can then be produced by any technique knownin the art, including recombinant DNA technology and/or chemicalsynthesis.

[0190] 3. Therapeutic Uses of the Fusion Molecules of the Invention

[0191] The present invention provides new therapeutic strategies fortreating immune diseases resulting from excess or inappropriate immuneresponse, as well as methods for the prevention of anaphylacticresponse. Specifically, the invention provides compounds and methods forthe treatment of type I hypersensitivity diseases mediated through thehigh-affinity IgE receptor, as well as for the treatment of autoimmunediseases (e.g., autoimmune diabetes mellitus, rheumatoid arthritis, andmultiple sclerosis). The invention provides advantages over existingmethods for treating immune diseases. The methods described herein finduse in the treatment of any mammalian subject, however, humans are apreferred subject.

[0192] Nature of the Diseases Targeted

[0193] Allergic reactions are classified following the Gell and CoombsClassification, depending on the type of immune response induced and theresulting tissue damage that develops as a result of reactivity to anantigen. A Type I reaction (immediate hypersensitivity) occurs when anantigen (called an allergen in this case) enters the body and encountersmast cells or basophils that are sensitized to the allergen as a resultof IgE specific to the allergen being attached to its high-affinityreceptor, FcεR1. Upon reaching the sensitized cell, the allergencross-links IgE molecules bound to FcεRI, causing an increase inintracellular calcium (Ca²⁺) that triggers the rapid release ofpre-formed mediators, such as histamine and proteases, and newlysynthesized, lipid-derived mediators such as leukotrienes andprostaglandins (i.e., degranulation). Excessive release of theseautocoids produces the acute clinical symptoms of allergy. Stimulatedbasophils and mast cells will also produce and release proinflammatorymediators, which participate in the acute and delayed phase of allergicreactions.

[0194] As discussed before and shown in Table 1 above, a large varietyof allergens has been identified so far, and new allergens areidentified, cloned and sequenced practically every day.

[0195] Ingestion of an allergen results in gastrointestinal and systemicallergic reactions. The most common food allergens involved are peanuts,shellfish, milk, fish, soy, wheat, egg and tree nuts such as walnuts. Insusceptible people, these foods can trigger a variety of allergicsymptoms, such as nausea, vomiting, diarrhea, urticaria, angioedema,asthma and full-blown anaphylaxis.

[0196] Inhalation of airborne allergens results in allergic rhinitis andallergic asthma, which can be acute or chronic depending on the natureof the exposure(s). Exposure to airborne allergens in the eye results inallergic conjunctivitis. Common airborne allergens includes pollens,mold spores, dust mites and other insect proteins. Cat, dust mite andcockroach allergens are the most common cause of perrenial allergicrhinitis while grass and weed and tree pollens are the most common causeof seasonal hay fever and allergic asthma.

[0197] Cutaneous exposure to an allergen, e.g. natural rubber latexproteins as found in latex gloves, may result in local allergicreactions manifest as hives (urticaria) at the places of contact withthe allergen. Absoprtion of the allergen via the skin may also causesystemic symptoms.

[0198] Systemic exposure to an allergen such as occurs with a bee sting,the injection of penicillin, or the use of natural rubber latex (NRL)gloves inside a patient during surgery may result in, cutaneous,gastrointestinal and respiratory reactions up to and including airwayobstruction and full blown anaphylaxis. Hymenoptera insect stings arecommonly cause allergic reactions, often leading the anaphylactic shock.Examples include various stinging insects including honeybees, yellowjackets, yellow hornets, wasps and white-faced hornets. Certain antsthat also sting known as fire ants (Solenopsis invicta) are anincreasing cause of serious allergy in the US as they expand their rangein this country. Proteins in NRL gloves have become an increasingconcern to health care workers and patients and at present, there is nosuccessful form of therapy for this problem except avoidance.

[0199] A large number of autoimmune diseases have also been identified,as well as the autoantigens recognized by the autoantibodies implicatedin the pathology of autoimmune diseases, as shown in Table 2, and knownin the art (see, e.g., van Venrooij and Maini (Eds.), Manual ofBiological Markers of Disease, Kluwer Academic Publishers [1996]; Roseand MacKay (Eds.), The Autoimmune Diseases, Third Edition, AcademicPress [1998]; and Lydyard and Brostoff (Eds.), Autoimmune Disease:Aetiopathogenesis, Diagnosis and Treatment, Blackwell Science Ltd.[1994]). The list of autoantigens and autoimmune diseases in Table 2 isnot exhaustive and is not intended to be limiting, as it is contemplatedthat new autoantigens and diseases with autoimmune etiologies will beidentified in the future. It is not intended that the invention belimited to the treatment of the diseases taught in Table 2, and it isnot intended that autoantigen sequences finding use with the inventionbe limited to those sequences provided in Table 2. Examples ofautoimmune diseases for which the autoantigen is not currently known,but may be identified in the future, includes but are not limited toBehcet's disease, Crohn's disease, Kawasaki's disease, autoimmune maleinfertility, Raynauds disease, Takayasu's arteritis and Giant cellarteritis.

[0200] Uses of Compounds for Targeted Diseases

[0201] The compounds disclosed herein can be used to treat or prevent alarge number of immune diseases, such as allergic diseases, autoimmunediseases, and anaphylactic shock response. The present inventionprovides new therapeutic strategies for treating immune diseasesresulting from excess or inappropriate immune response. Specifically,the invention provides compositions and methods finding the usesdescribed below. The uses itemized herein are not intended to belimiting, as modification of these uses will be apparent to one familiarwith the art.

[0202] (a) The invention finds use in the treatment of type Ihypersensitivity diseases mediated through the high-affinity IgEreceptor (e.g., allergic diseases, such as allergic asthma). In thesemethods, the FcεR receptors are crosslinked to inhibitory FcγR receptorsvia the fusion polypeptides of the present invention, resulting in adownregulation of the IgE and FcεR activity. The compounds disclosedherein can be used to inhibit or prevent acute or chronic IgE mediatedreactions to major environmental and occupational allergens.

[0203] When the fusion polypeptide compositions of the present inventioncomprise IgG heavy chain constant region sequences and allergensequences, the immune suppression will be specific for the particularallergen. When the fusion polypeptide compositions of the presentinvention comprise IgG heavy chain constant region sequences and IgEheavy chain constant region sequences, the suppression of the type Ihypersensitivity response will be global, and not specific for aparticular allergen.

[0204] (b) Some fusion polypeptide compositions of the invention can beused to provide vaccination material suitable for allergy immunotherapyto induce a state of non-allergic reactivity (i.e., desensitisation orallergic tolerance) to specific allergens. When used in this capacity,the fusion polypeptide material comprises IgG heavy chain constantregion sequences and allergen sequences. It is contemplated that in thiscase, the fusion polypeptide is internalized, processed and presented onthe surface of cells (e.g., but not limited to APCs). Use of the fusionpolypeptides in this manner provide an advantage over existingvaccination materials, as the fusion polypeptide has intrinsic abilityto prevent or downregulate any acute type I hypersensitivity response(e.g., an anaphylactic reaction) that may result from response to theallergen sequence component of the fusion polypeptide. It iscontemplated that this prevention or downregulation occurs throughcrosslinking of the stimulatory Fcε receptors with inhibitory Fcγreceptors via the fusion polypeptide and endogenous IgE specific for theallergen sequence. However, it is not necessary to understand themechanism responsible for the downregulation in order to make or use thepresent invention. In this embodiment, the fusion polypeptide may or maynot comprise particular amino acid sequences that promote targetting andproteolytic processing that facilitate copresentation of the antigensequence with MHC I or MHC II for the induction of tolerance.

[0205] (c) Some fusion polypeptide compositions of the inventioncomprising IgG heavy chain constant region sequences and autoantigensequences (e.g., myelin basic protein) find use in the treatment ofautoimmune diseases (e.g., multiple sclerosis) as vaccination materialsuitable for use in immunotherapy. When used in this capacity, it iscontemplated that the polypeptide material is processed and presented onantigen presenting cells (APCs). In this embodiment, the fusionpolypeptide may or may not comprise particular amino acid sequences thatpromote targetting and proteolytic processing that facilitatecopresentation of the autoantigen sequence with MHC I or MHC II for theinduction of tolerance. The fusion polypeptide material used in thismode of therapy has the additional benefit of having the intrinsicability to prevent or downregulate any acute type I hypersensitivityresponse (e.g., an anaphylactic reaction) that may result fromreactivity directed against the autoantigen component on the fusionpolypeptide. It is contemplated that this downregulation occurs throughcrosslinking the stimulatory Fcε receptors with inhibitory Fcγ receptorsvia the fusion polypeptide and endogenous IgE specific for theautoantigen sequence. However, it is not necessary to understand themechanism responsible for the downregulation in order to make or use thepresent invention.

[0206] (d) The fusion polypeptides of the present invention can be usedin conjunction with traditional whole antigen desensitization or peptideimmunotherapies in the treatment of allergies or autoimmune disorders,for the purpose of preventing the dangerous anaphylactic reactionsfrequently observed in response to traditional immunotherapies. Whenused in this capacity, the fusion polypeptide compositions of theinvention will comprise IgG heavy chain constant region sequences, aswell as either IgE heavy chain constant region sequences, allergenpeptide sequences, or autoantigen peptide sequences. It is contemplatedthat the fusion polypeptide can be delivered to a subject before, duringor after the delivery of other traditional peptide therapies in thetreatment of allergic or autoimmune diseases to prevent anaphylacticreaction in response to the immunotherapy material. In a preferredembodiment, the fusion polypeptide composition can be given to a subjectwho has previously displayed type I hypersensitivity to a particularwhole antigen or peptide during immunotherapy, and thus, is at risk forhypersensitivity responses to future immunotherapies with that sameantigen. This use of the fusion polypeptides of the invention willprovide a platform for the reinstitution of traditional peptidetherapies that were previously abandoned due to their induction ofsystemic hypersensitivity effects (e.g., causing anaphylacticreactions).

[0207] (e) The compositions and methods of the invention can provide aprophylactic effect against allergic disease by preventing allergicsensitization to environmental and occupational allergens whenadministered to at-risk individuals (e.g., those at genetic risk ofasthma and those exposed to occupational allergens in the workplace).

[0208] (f) It is contemplated that the methods for treating a subjectusing the fusion polypeptides of the invention may comprise thesimultaneous delivery of more than one fusion polypeptide to achieve adesired curative or prophylactic effect. For example, an allergen orautoantigen may not have a single immunodominant epitope, andalternatively, may have multiple epitopes recognized by native IgEmolecules. In that case, multiple fusion polypeptides, each comprising adifferent epitope, can be delivered to a subject.

[0209] In another example, patients who demonstrate an autoimmunedisorder frequently test positive for the presence of more than one typeof autoantibody, and thus, have more than one physiological autoantigen.In that case, it is contemplated that the methods for treating thatpatient may comprise the simultaneous delivery of more than one fusionpolypeptide to achieve the desired immunosuppressive effect, where eachfusion polypeptide comprises a different suitable autoantigen sequence.In this case, the fusion polypeptide(s) can also be givenprophylactically, for the purpose of preventing the anaphylacticresponses that may occur during autoantigen tolerance therapy.

[0210] (g) It is also contemplated that in some embodiments of theinvention, the fusion polypeptides are used in combination with othertreatments, e.g., co-delivery with biological modifiers (e.g.,antagonists of inflammatory response mediators, including tumor necrosisfactor a (TNFα), IL-1, IL-2, interferon-α (INF-α), and INF-β),immuno-suppressive therapy (e.g., methotrexate, calcineurin inhibitorsor steroids), or various adjuvants, as known in the art.

[0211] Advantages of the Invention

[0212] The bifunctional gamma-epsilon compounds (i.e., the fusionpolypeptides) described can be used to prevent allergic reactions to anyspecific allergen or group of allergens. By occupying a critical numberof FcεRI receptors, these molecules will inhibit the ability ofbasophils and mast cells to react to any allergen so as to preventincluding, without limitation, asthma, allergic rhinitis, atopicdermatitis, food allergies, forms of autoimmune urticaria andangioedema, up to and including anaphylactic shock. Thus these compoundscould be used acutely to desensitize a patient so that theadministration of a therapeutic agent (e.g., penicillin) can be givensafely. Similarly, they can be used to desensitize a patient so thatstandard allergen vaccination may be given with greater safety, e.g.,peanut or latex treatment. They can also be used as chronic therapy toprevent clinical reactivity to prevent environmental allergens such asfoods or inhalant allergens.

[0213] The present invention provides gamma-allergen bifunctional fusionmolecules for use in a novel form of allergy vaccination that will besafer and more effective in the treatment of a variety of IgE-mediatedallergic reactivity, including, without limitation, asthma, allergicrhinitis, atopic dermatitis, food allergies, urticaria and angioedema,up to and including anaphylactic shock. Having the allergen fused to amolecule that will bind to FcγRIIb on mast cells and basophils willprevent the allergen from inducing local or systemic allergic reactions.Such local or systemic allergic reactions are major problem in allergenvaccination as currently practiced. The gamma-allergen fusion proteinswill be able to be given in higher doses over a shorter interval andwith greater safety than standard allergen therapy. These benefits ofthe invention are equally applicable to the situation where delivery ofa traditional vaccine for the treatment of an autoimmune disease maycause a severe IgE-mediated (i.e., allergic) immune response, includinganaphylactic shock.

[0214] In addition, use of the gamma-allergen compounds will causeantigen specific desensitization to that specific allergen. Thus thegamma-allergen compounds will give a window of safe exposure to theallergen be it as an acute or recurring treatment as would be needed inusing a therapeutic monoclonal antibody to which a patient has developedan allergic (IgE) response or as chronic treatment for prevention ofunintentional exposures such as occurs with peanut allergens.

[0215] The importance of being able to suppress a hypersensitivityresponse is expected to increase with the development of recombinant DNAand protein technologies. As an increasing number of recombinantpolypeptide products find their way into therapeutic applications in thenear future, there is an increased likelihood that these recombinantproducts will trigger hyperimmune responses. The gamma-allergencompounds can even be used along with conventional allergen vaccinationso as to provide an extra margin of safety while large doses of standardallergen are given. Similarly, the fusion polypeptides of the presentinvention can be used in conjunction with recombinant polypeptidetherapeutics so as to diminish the risk of hyperimmune response to therecombinant therapeutic.

[0216] The bifunctional autoantigen-Fcγ fusion polypeptides describedcan be used prophylactically to prevent type-I hypersensitivityreactions to autoantigen sequences used in autoantigen tolerance therapyfor the treatment of autoimmune disease. It is contemplated that acritical number of Fcε and inhibitory Fcγ receptors will be crosslinkedvia the formation of a bridge comprising the fusion polypeptide andendogenous IgE specific for the autoantigen sequence (however, it is notnecessary to understand the mechanisms of immune suppression to make oruse the invention). Thus, these fusion polypeptides will inhibit theability of basophils and mast cells to react to exogenously suppliedautoantigen, as would be encountered during tolerance therapy, so as toprevent type-I hypersensitivity reactions, up to and includinganaphylactic shock. These fusion polypeptides could be used todesensitize a patient so that the therapeutic administration ofautoantigen peptide (i.e., the tolerance therapy) can take place withgreater safety.

[0217] The present invention provides autoantigen-Fcγ fusionpolypeptides for use in a novel form of autoimmune vaccination that willbe safer and more effective in the treatment of autoimmune disease. Thefusion polypeptide can be coadminstered with isolated autoantigen, oralternatively, no supplemental autoantigen is administered. Having theautoantigen sequence fused to a molecule that will bind to FcγRIIb onmast cells and basophils will prevent the autoantigen sequence (eitherby itself or as part of the fusion polypeptide) being able to inducelocal or systemic type I hypersensitivity reactions. Such local orsystemic allergic reactions are a major concern in vaccination therapiesas currently practiced. The fusion polypeptides comprising autoantigenand Fcγ will permit the administration of autoantigen sequences inhigher doses over a shorter interval and with greater safety thanstandard autoantigen-alone peptide therapy.

[0218] Alternatively, when used in conjunction with free autoantigen, afusion polypeptide comprising Fcε and Fcγ can be used during thedesensitization therapy, for the purpose of suppressing type-Ihypersensitivity reactions. This Fcε-Fcγ fusion polypeptide has theadded advantage that it can be used to suppress any IgE-mediated type-Ihypersensitivity response, and not only the response solicited from aparticular autoantigen sequence.

[0219] Furthermore, use of the autoantigen-Fcγ fusion compounds willresult in antigen specific suppression (i.e., desensitization) to thatspecific autoantigen. This antigen-specific immune suppression isstrongly preferable to generalized immune suppression, as broadsuppression leaves the patient susceptible to possibly life-threateninginfections (in addition to the side effects of the potentimmunosuppressive drugs, such as cyclosporine A and methotrexate).

[0220] In addition, the chimeric gamma-epsilon compounds herein holdgreat promise for the treatment of autoimmune chronic urticaria andangioedema. Urticaria is a skin symptom that may accompany allergies butoften is idiopathic. It is a relatively common disorder caused bylocalized cutaneous mast cell degranulation, with resultant increaseddermal vascular permeability culminating in pruritic wheals. Angioedemais a vascular reaction involving the deep dermis or subcutaneous orsubmucosal tissues caused by localized mast cell degranulation. Thisresults in tissue swelling that is pruritic or painful. Chronicurticaria and angioedema often occur together although they occurindividually as well. These conditions are common and once present formore than six months, they often last a decade or more. Although notfatal, they are very troubling to patients, as the frequency ofrecurring attacks disrupts daily activities and thereby results insignificant morbidity. Standard therapy is often unsuccessful in theseconditions, and is distressing to the point that chemotherapy withcyclosporine A and other potent immunosuppressive drugs has recentlybeen advocated. Increasing evidence suggests that as many as 60% ofpatients with these conditions actually have an autoimmune disease, inwhich they make functional antibodies against the FcεRI receptor. Forfurther details, see Hide et al., N. Engl. J. Med. 328:1599-1604 (1993);Fiebiger et al., J. Clin. Invest. 96:2606-12 (1995); Fiebiger et a., J.Clin. Invest. 101:243-51 (1998); Kaplan, A. P., Urticaria andAngioedema, In: Inflammation: Basic Principles and Clinical Correlates(Galliin and Snyderman eds.), 3^(rd) Edition, Lippincott & Wilkins,Philadelphia, 1999, pp. 915-928. The fusion molecules of the presentinvention are believed to form the basis for a novel and effectivetreatment of these diseases by safely blocking access to the FcεRI.

[0221] Compositions and Formulations of the Invention

[0222] For therapeutic uses, including prevention, the compounds of theinvention can be formulated as pharmaceutical compositions in admixturewith pharmaceutically acceptable carriers or diluents. Methods formaking pharmaceutical formulations are well known in the art. Techniquesand formulations generally may be found in Remington's PharmaceuticalSciences, 18th Edition, Mack Publishing Co., Easton, Pa. 1990. See,also, Wang and Hanson “Parenteral Formulations of Proteins and Peptides:Stability and Stabilizers”, Journal of Parenteral Science andTechnology, Technical Report No. 10, Supp. 42-2S (1988). A suitableadministration format can best be determined by a medical practitionerfor each patient individually.

[0223] Pharmaceutical compositions of the present invention can comprisea fusion molecule of the present invention along with conventionalcarriers and optionally other ingredients.

[0224] Suitable forms, in part, depend upon the use or the route ofentry, for example oral, transdermal, inhalation, or by injection. Suchforms should allow the agent or composition to reach a target cellwhether the target cell is present in a multicellular host or inculture. For example, pharmacological agents or compositions injectedinto the blood stream should be soluble. Other factors are known in theart, and include considerations such as toxicity and forms that preventthe agent or composition from exerting its effect.

[0225] Carriers or excipients can also be used to facilitateadministration of the compound. Examples of carriers and excipientsinclude calcium carbonate, calcium phosphate, various sugars such aslactose, glucose, or sucrose, or types of starch, cellulose derivatives,gelatin, vegetable oils, polyethylene glycols and physiologicallycompatible solvents. The compositions or pharmaceutical composition canbe administered by different routes including, but not limited to, oral,intravenous, intra-arterial, intraperitoneal, subcutaneous, intranasalor intrapulmonary routes.

[0226] The desired isotonicity of the compositions can be accomplishedusing sodium chloride or other pharmaceutically acceptable agents suchas dextrose, boric acid, sodium tartrate, propylene glycol, polyols(such as mannitol and sorbitol), or other inorganic or organic solutes.

[0227] For systemic administration, injection is preferred, e.g.,intramuscular, intravenous, intra-arterial, etc. For injection, thecompounds of the invention are formulated in liquid solutions,preferably in physiologically compatible buffers such as Hank's solutionor Ringer's solution. Alternatively, the compounds of the invention areformulated in one or more excipients (e.g., propylene glycol) that aregenerally accepted as safe as defined by USP standards. They can, forexample, be suspended in an inert oil, suitably a vegetable oil such assesame, peanut, olive oil, or other acceptable carrier. Preferably, theyare suspended in an aqueous carrier, for example, in an isotonic buffersolution at pH of about 5.6 to 7.4. These compositions can be sterilizedby conventional sterilization techniques, or can be sterile filtered.The compositions can contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH buffering agents. Useful buffers include for example, sodiumacetate/acetic acid buffers. A form of repository or “depot” slowrelease preparation can be used so that therapeutically effectiveamounts of the preparation are delivered into the bloodstream over manyhours or days following transdermal injection or delivery. In addition,the compounds can be formulated in solid form and redissolved orsuspended immediately prior to use. Lyophilized forms are also included.

[0228] Alternatively, certain molecules identified in accordance withthe present invention can be administered orally. For oraladministration, the compounds are formulated into conventional oraldosage forms such as capsules, tablets and tonics.

[0229] Systemic administration can also be by transmucosal ortransdermal. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, bile salts and fusidic acidderivatives. In addition, detergents can be used to facilitatepermeation. Transmucosal administration can be, for example, throughnasal sprays or using suppositories.

[0230] A preferred route for administration of the compounds of theinvention may be inhalation for intranasal and/or intrapulmonarydelivery. For administration by inhalation, usually inhalable dry powercompositions or aerosol compositions are used, where the size of theparticles or droplets is selected to ensure deposition of the activeingredient in the desired part of the respiratory tract, e.g. throat,upper respiratory tract or lungs. Inhalable compositions and devices fortheir administration are well known in the art. For example, devices forthe delivery of aerosol medications for inspiration are known. One suchdevice is a metered dose inhaler that delivers the same dosage ofmedication to the patient upon each actuation of the device. Metereddose inhalers typically include a canister containing a reservoir ofmedication and propellant under pressure and a fixed volume metered dosechamber. The canister is inserted into a receptacle in a body or basehaving a mouthpiece or nosepiece for delivering medication to thepatient. The patient uses the device by manually pressing the canisterinto the body to close a filling valve and capture a metered dose ofmedication inside the chamber and to open a release valve which releasesthe captured, fixed volume of medication in the dose chamber to theatmosphere as an aerosol mist. Simultaneously, the patient inhalesthrough the mouthpiece to entrain the mist into the airway. The patientthen releases the canister so that the release valve closes and thefilling valve opens to refill the dose chamber for the nextadministration of medication. See, for example, U.S. Pat. No. 4,896,832and a product available from 3M Healthcare known as Aerosol SheathedActuator and Cap.

[0231] Another device is the breath actuated metered dose inhaler thatoperates to provide automatically a metered dose in response to thepatient's inspiratory effort. One style of breath actuated devicereleases a dose when the inspiratory effort moves a mechanical lever totrigger the release valve. Another style releases the dose when thedetected flow rises above a preset threshold, as detected by a hot wireanemometer. See, for example, U.S. Pat. Nos. 3,187,748; 3,565,070;3,814,297; 3,826,413; 4,592,348; 4,648,393; 4,803,978.

[0232] Devices also exist to deliver dry powdered drugs to the patient'sairways (see, e.g. U.S. Pat. No. 4,527,769) and to deliver an aerosol byheating a solid aerosol precursor material (see, e.g. U.S. Pat. No.4,922,901). These devices typically operate to deliver the drug duringthe early stages of the patient's inspiration by relying on thepatient's inspiratory flow to draw the drug out of the reservoir intothe airway or to actuate a heating element to vaporize the solid aerosolprecursor.

[0233] Devices for controlling particle size of an aerosol are alsoknown, see, for example, U.S. Pat. Nos. 4,790,305; 4,926,852; 4,677,975;and 3,658,059.

[0234] For topical administration, the compounds of the invention areformulated into ointments, salves, gels, or creams, as is generallyknown in the art.

[0235] If desired, solutions of the above compositions can be thickenedwith a thickening agent such as methyl cellulose. They can be preparedin emulsified form, either water in oil or oil in water. Any of a widevariety of pharmaceutically acceptable emulsifying agents can beemployed including, for example, acacia powder, a non-ionic surfactant(such as a Tween), or an ionic surfactant (such as alkali polyetheralcohol sulfates or sulfonates, e.g., a Triton).

[0236] Compositions useful in the invention are prepared by mixing theingredients following generally accepted procedures. For example, theselected components can be mixed simply in a blender or other standarddevice to produce a concentrated mixture which can then be adjusted tothe final concentration and viscosity by the addition of water orthickening agent and possibly a buffer to control pH or an additionalsolute to control tonicity.

[0237] The amounts of various compounds for use in the methods of theinvention to be administered can be determined by standard procedures.Generally, a therapeutically effective amount is between about 100 mg/kgand 10-12 mg/kg depending on the age and size of the patient, and thedisease or disorder associated with the patient. Generally, it is anamount between about 0.05 and 50 mg/kg, more preferably between about1.0 and 10 mg/kg for the individual to be treated. The determination ofthe actual dose is well within the skill of an ordinary physician.

[0238] The compounds of the present invention may be administered incombination with one or more further therapeutic agent for the treatmentof IgE-mediated allergic diseases or conditions. Such furthertherapeutic agents include, without limitation, corticosteroids,β-antagonists, theophylline, leukotriene inhibitors, allergenvaccination, soluble recombinant human soluble IL-4 receptors(Immunogen), anti-IL-4 monoclonal antibodies (Protein Design Labs), andanti-IgE antibodies, such as the recombinant human anti-IgE monoclonalantibody rhuMAb-E25 (Genentech, Inc.) which is currently in advancedclinical trials for the treatment of patients with atopic asthma, andother allergic diseases, such as allergic rhinitis and atopic dermatitis(see, e.g. Barnes, The New England Journal of Medicine 341:2006-2008(1999)). Thus the compounds of the present invention can be used tosupplement traditional allergy therapy, such as corticosteroid therapyperformed with inhaled or oral corticosteroids.

[0239] 4. Articles of Manufacture

[0240] The invention also provides articles of manufacture comprisingthe single-chain fusion compounds herein. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, etc. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is effective for treating the condition and may have a sterileaccess port (for example the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). The container may also be an inhalation device such as thosediscussed above. At least one active agent in the composition is afusion compound of the invention. The label or package insert indicatesthat the composition is used for treating the condition of choice, suchas an allergic condition, e.g., asthma or any of the IgE-mediatedallergies discussed above. The article of manufacture may furthercomprise a further container comprising a pharmaceutically-acceptablebuffer, such as bacteriostatic water for injection (BWFI),phosphate-buffered saline, Ringer's solution and dextrose solution. Itmay further include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles, andsyringes.

[0241] Further details of the invention are illustrated by the followingnon-limiting Examples.

EXAMPLE 1

[0242] Construction and Expression of a Chimeric Human Fcγ-Fcε FusionProtein

[0243] Materials and Methods

[0244] Plasmids, Vectors and Cells

[0245] Plasmid pAG 4447 containing genomic DNA encoding human IgEconstant region and expression vector pAN 1872 containing human genomicDNA encoding the hinge-CH2—CH3 portion of IgG₁ constant region wereobtained from the laboratory of Dr. Morrison. pAN 1872 is derived fromthe pDisplay vector (Invitrogen). pAG 4447 was developed and used as acloning intermediate in the construction of a human IgE expressionvector disclosed in J. Biol. Chem. 271:3428-3436 (1996). To constructthe chimeric gene, a pair of primers were designed to amplify the humanIgE constant region (CH2-CH3-CH4). 5′-end primer: (SEQ ID NO:8)5′GCTCGAGGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGTTCACCCCGCCCACCGTGAAG3′,

[0246] containing a flexible linker sequence and an XhoI site.

[0247] 3′ end primer:

[0248] 5′GGCGGCCGCTCATTTACCGGGATTTACAGACAC3′ (SEQ ID NO: 9),

[0249] containing a NotI site.

[0250] After amplification, the PCR products were cloned into pCR2.1vector (Invitrogen). The sequences of the products were confirmed. Then,the ZhoI-NotI fragment was inserted into the 1782 pAN vector, followingthe IgG₁ CH3 domain in the same reading frame by a (Gly₄Ser)₃ flexiblelinker. SP2.0 murine myeloma cell line was selected as host forexpression because it does not secrete any antibody.

[0251] Expression and Purification

[0252] The expression vector containing chimeric Fcγ-Fcε gene waslinearized at the PvuI site and transfected into SP2/0 cells byelectroporation (Bio-Rad). Stable transfectants were selected for growthin medium containing 1 mg/ml geneticin. Clones producing the fusionprotein were identified by ELISA using plates coating anti-human IgE(CIA7.12) or IgG (Sigma) antibody. Supernatants from clones were addedto wells, and bound protein was detected using goat anti-human IgE orIgG conjugated to alkaline phosphatase (KPL). The fusion protein waspurified from the supernatants and ascites by using rProtein A column(Pharmacia).

[0253] Western Blotting

[0254] The purified protein was run on 7.5% SDS polyacrylamide gel.After transfer, the nylon membrane was blocked by 4% bovine serumalbumin/PBS/Tween overnight at 4° C. For protein detection, the blot wasprobed with either goat anti-human IgE (ε chain specific) or goatanti-human IgG (γ chain-specific) conjugated to alkaline phosphatase(KPL). Color development was performed with an alkaline phosphataseconjugated substrate kit (Bio-Rad).

[0255] Binding Test

[0256] In order to confirm the binding, FcεRI transfected cells (CHO3D10) or human HMC-1 cells that express FcγRIIb but not FcεRI werestained with purified fusion protein and then analyzed by flowcytometry. Briefly, cells were collected and washed. The cells were thenincubated with 5 μl of 1 mg/ml GE2, PS IgE or human IgG at 4° C. for 60minutes. After two washes, the cells were stained with FITC conjugatedanti-human IgE or IgG at 4° C. for 60 minutes, and visualized by flowcytometry.

[0257] Inhibition of Basophil Histamine Release

[0258] Acid-stripped Percoll-enriched human blood basophils were primedwith 1-10 μg/ml of chimeric human anti-NP IgE at 37° C. in a 5% CO₂incubator and one hour later, challenged with 30 ng of NP-BSA (Kepley,J. Allergy Clin. Immunol. 106:337-348(2000)). Histamine release wasmeasured in the supernatants 30 minutes later. GE2 or control humanmyeloma IgE was added at various doses and times to test the effects onhistamine release.

[0259] Passive Cutaneous Anaphylaxis Model

[0260] Transgenic mice expressing the human FcεRIα chain and with themurine FcεRIα chain knocked out (provided by Dr. Jean-Pierre Kinet,Harvard Medical School, Boston, Mass., Dombrowicz, et al, J. Immunol.157:1645-1654. (1996)) were primed cutaneously with either recombinanthuman anti-dansyl or anti-NP IgE. Individual sites were then injectedwith saline, GE2 or IgE myeloma protein. Four hours later, mice weregiven a systemic challenge with dansyl-OVA or NP-BSA plus Evans blue,and the resulting area of reaction was measured.

[0261] Results

[0262] Western blotting showed that the chimeric protein (designatedGE2) was expressed as the predicted dimer of approximately 140 kD. TheGE2 protein reacted with both anti-human ε and anti-human γchain-specific antibodies.

[0263] GE2 showed the ability to inhibit IgE-mediated release ofhistamine from fresh human basophils. The results of the dose-dependentinhibition of basophil histamine release using the fusion protein GE2(±SEM; n+3 separate donors, each in duplicate) are shown in FIG. 8. Thedata show that, when added to fresh human basophils along with thesensitizing anti-NP IgE antibody, GE2 inhibited subsequent NP-inducedrelease of histamine in a dose-dependent manner, more effectively thanan equivalent amount of native human IgE protein. This was timedependent as expected with the greatest effect being observed when theGE2 was added with the sensitizing anti-NP IgE antibody. No effect wasobserved if the GE2 was given simultaneously with the antigen challenge.

[0264] To test the in vivo function of GE2, the transgenic passivecutaneous anaphylaxis described above was used. The results are shown inFIG. 9. The size and color of the reaction at the sites of GE2 injectionwere decreased compared to those injected with comparable amount ofhuman IgE.

[0265] These results demonstrate that the GE2 protein is able to inhibitmast cell/basophil function greater than an equivalent amount of IgE andimplicates binding to both FcεR1 and FCγ R.

[0266] Analysis of binding using flow cytometry showed that the GE2protein bound in a fashion similar to native IgE to the human FcγR11expressed on HMC-1 cells. The data are shown in FIG. 10. Similar resultswere obtained for the FcεRI on 3D10 cells, as shown in FIG. 11.

EXAMPLE 2

[0267] Construction and Expression of Chimeric Human Fcε-autoantigenFusion Proteins for Use in

[0268] Treating Subjects with Multiple Sclerosis

[0269] Two human F_(c)γ-autoantigen fusion polypeptides are producedusing recombinant DNA techniques and a mammalian protein overexpressionsystem. The resulting recombinant fusion proteins are purified usingimmunoprecipitation techniques and analyzed, as described below. Twoforms of the fusion polypeptide are described. Both forms of the fusionpolypeptide contain the hinge-CH2-CH3 portion of the IgG₁ constantregion, as provided in SEQ ID NO: 1. One form of the fusion polypeptidecomprises a full length myelin-basic-protein (MBP) amino acid sequence(as provided in SEQ ID NO:12), while an alternative version of thefusion polypeptide comprises a portion of MBP containing essentially theminimal, immunodominant autoimmune epitope, i.e., MBP₈₃₋₉₉.(Warren etal., Proc. Natl. Acad. Sci. USA 92:11061-11065 [1995] and Wucherpfenniget al., J. Clin. Invest., 100(5):1114-1122 [1997]). This minimal MBPepitope has the amino acid sequence:

[0270] E₈₃NPVVHFFKNIVTPRTP₉₉ (SEQ ID NO: 13)

[0271] The resulting fusion polypeptides find use in the treatment ofautoimmune multiple sclerosis, as well as for the prevention ofanaphylactic response which may result from exposure to exogenous MBPpolypeptide, as would be encountered during tolerance therapy.

[0272] Vectors

[0273] Mammalian expression vectors encoding the fusion polypeptides areconstructed by subcloning the IgG and MBP autoantigen sequences into asuitable vector. In this Example, a modified form of the pDisplay vector(Invitrogen) is used as the backbone, called pAN1872, which uses theconstitutively active PCMV promoter to transcribe subcloned sequences,and produces these sequences with an in-frame hemagglutinin (HA) epitopetag. The modified vector encodes a secreted form of the subclonedsequences. The pAN1872 vector contains human genomic DNA encoding thehinge-CH2-CH3 portion of IgG₁ constant region, as described in Example 1and SEQ ID NO: 1.

[0274] To construct the chimeric IgG-autoantigen expression vector,myelin-basic-protein (MBP) sequences are amplified from an MBP cDNAvector using PCR protocols. Any vector containing MBP cDNA sequence canbe a suitable template for the PCR reaction. The PCR primers aredesigned to permit the amplification of the full length MBP cDNA, oralternatively, any suitable portion of the MBP cDNA. The PCR primersused are not limited to a particular nucleotide sequence, as variousprimers can be used dependent on variations in the template backbone andthe desired MBP portion(s) for amplification.

[0275] The resulting double stranded PCR products are then subclonedinto the pANI 872 vector, in such a way that the coding sequences of IgGheavy chain constant region and the MBP sequences are in frame toproduce a single translation product. The suitable PCR primers can alsobe designed to incorporate a flexible linker sequence (e.g., [Gly₄Ser]₃)and terminal endonuclease restriction sites to facilitate the in-framesubcloning, and are further designed to permit the subcloning of the MBPsequences at the carboxy-terminus (C-terminus) of the IgG heavy chainconstant region.

[0276] A portion of MBP as small as the MBP₈₃₋₉₉ immunodominant epitopealso finds use with the present invention. In this case, a suitabledouble-stranded oligonucleotide can be generated using synthetic meansfor use in the subcloning step. The nucleotide sequence of theengineered fusion construct coding sequences is confirmed by DNAsequencing.

[0277] Expression and Purification

[0278] Following construction of the mammalian expression vectors above,these vectors are linearized by single-site cleavage with a suitablerestriction enzyme (e.g., PvuI). These linearized nucleic acids are thentransfected in the SP2.0 cell line (a murine myeloma) using anelectroporation apparatus and reagents (Bio-Rad). The SP2.0 cell line isused, as it does not secrete antibody, and will not contaminate thepurified antibody encoded by the transfected expression vector.

[0279] Following the electroporation, stable transfectants are selectedin Iscove's modified Dulbecco's growth medium supplemented with 1 mg/mlgeneticin. Supernatants from surviving clones are collected and analyzedfor fusion molecule production by ELISA, using plates coated with rabbitanti-IgG antibody (Sigma). The fusion molecules are then specificallydetected using a goat anti-human IgG conjugated to alkaline phosphatase(KPL) detection antibody. SP2.0 clones producing the fusion molecule arethus identified.

[0280] Purification

[0281] The fusion polypeptide contained in the SP2.0 cell culturesupernatants is purified using rProtein A column purification(Pharmacia). Alternatively, as a source of starting material for thepurification, the SP2.0 cell lines is used to produce ascites fluid innude mice. The ascites fluid is collected and purified using rProtein Acolumn purification. Alternatively still, the fusion polypeptide ispurified from cell culture supernatants or ascites fluids using ananti-HA immunoaffinity purification, as the fusion polypeptides aretranslated with an in-frame hemagglutinin tag encoded by the pDisplayvector. Such purification methods are well known in the art.

[0282] Western Blotting

[0283] The fusion polypeptide is analyzed by Western immunoblottinganalysis. The purified polypeptide material is run on a 7.5% SDSpolyacrylamide gel. Following transfer to nylon membrane, the blot isblocked using 4% bovine serum albumin/PBS/Tween overnight at 4° C. Forprotein detection, the blot is probed with goat anti-human IgG (γchain-specific) conjugated to alkaline phosphatase (KPL). Colordevelopment is performed with an alkaline phosphatase-conjugatedsubstrate kit (Bio-Rad). Alternatively, anti-HA antibodies can be usedas the primary detection antibody in the Western blot.

[0284] Binding Test

[0285] In order to confirm the binding of the fusion polypeptide toFcγ-receptors, human HMC-1 cells that express FcγRIIb are contacted withpurified fusion protein and then analyzed by flow cytometry. Briefly,cells are collected, washed, then incubated with 5 μl of 1 mg/ml fusionpolypeptide, or alternatively, with human IgG at 4° C. for 60 minutes.After two washes, the cells are stained with FITC-conjugated anti-humanIgG at 4° C. for 60 minutes, and visualized by flow cytometry.

[0286] Inhibition of Basophil Histamine Release

[0287] The ability of the fusion polypeptide to suppress histaminerelease is assessed using a histamine release assay. Acid-strippedPercoll-enriched human blood basophils are primed with 1-10 μg/ml ofchimeric human anti-NP IgE at 37° C. in a 5% CO₂ incubator and one hourlater, and challenged with 30 ng of NP-BSA (Kepley, J. Allergy Clin.Immunol. 106:337-348(2000)). Histamine release is measured in thesupernatants 30 minutes later. Fusion polypeptide or control humanmyeloma IgE are added at various doses and times to test the effects onhistamine release.

[0288] Passive Cutaneous Anaphylaxis Model

[0289] The ability of the fusion polypeptide to suppress anaphylaxis isassessed using a mouse model assay. Transgenic mice expressing the humanFcεR1α chain and with the murine FcεRIα chain knocked out (provided byDr. Jean-Pierre Kinet, Harvard Medical School, Boston, Mass.,Dombrowicz, et al, J. Immunol. 157:1645-1654. (1996)) are primedcutaneously with either recombinant human anti-dansyl or anti-NP IgE.Individual sites are then injected with saline, fusion polypeptide orIgE myeloma protein. Four hours later, mice are given a systemicchallenge with dansyl-OVA or NP-BSA plus Evans blue, and the resultingarea of reaction is measured.

[0290] All references cited throughout the specification are herebyexpressly incorporated by reference. It is understood that theapplication of the teachings of the present invention to a specificproblem or situation will be within the capabilities of one havingordinary skill in the art in light of the teachings contained herein.Examples of the products of the present invention and representativeprocesses for their production and use should not be construed to limitthe invention.

What is claimed is:
 1. An isolated fusion molecule comprising a firstpolypeptide sequence capable of specific binding to a native IgGinhibitory receptor comprising an immune receptor tyrosine-basedinhibitory motif (ITIM), functionally connected to a second polypeptidesequence capable of specific binding, through a third polypeptidesequence, to a native IgE receptor (FcεR), wherein said first and secondpolypeptide sequences are other than antibody variable region sequencesand wherein said fusion molecule is not capable of T cell interactionprior to internalization.
 2. The fusion molecule of claim 1 wherein saidsecond polypeptide sequence comprises an antigen sequence.
 3. The fusionmolecule of claim 2 wherein said antigen sequence is at least a portionof an autoantigen sequence.
 4. The fusion molecule of claim 3 whereinsaid the autoantigen sequence comprises at least one autoantigenicepitope.
 5. The fusion molecule of claim 3, wherein said thirdpolypeptide is an immunoglobulin specific for said autoantigen sequence.6. The fusion molecule of claim 5, wherein said immunoglobulin specificfor said autoantigen sequence is an IgE class antibody.
 7. The fusionmolecule of claim 3 wherein said autoantigen sequence is selected fromthe group consisting of rheumatoid arthritis autoantigen, multiplesclerosis autoantigen, autoimmune type I diabetes mellitus autoantigen,and portions thereof.
 8. The fusion molecule of claim 7 wherein saidautoantigen is selected from the group consisting of myelin basicprotein (MBP), proteolipid protein, myelin oligodendrocyte glycoprotein,αβ-crystallin, myelin-associated glycoprotein, Po glycoprotein, PMP22,2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNPase), glutamic aciddecarboxylase (GAD), insulin, 64 kD islet cell antigen (IA-2, alsotermed ICA512), phogrin (IA-2β), type II collagen, human cartilage gp39(HCgp39), and gp130-RAPS.
 9. The fusion molecule of claim 3 wherein saidautoantigen sequence present in said fusion molecule has at least 90%sequence identity with at least a portion of a native autoantigensequence.
 10. The fusion molecule of claim 3 wherein said autoantigensequence present in said fusion molecule is encoded by a nucleic acidhybridizing under stringent conditions to the complement of a nucleicacid molecule encoding a native autoantigen.
 11. The fusion molecule ofclaim 3 wherein said inhibitory receptor is a low-affinity FcγRIIb IgGreceptor.
 12. The fusion molecule of claim 11 wherein said IgE receptoris a high-affinity FcεRI IgE receptor.
 13. The fusion molecule of claim11 wherein said IgE receptor is a low-affinity FcεRII (CD23) IgEreceptor.
 14. The fusion molecule of claim 12 wherein said FcγRIIb andFcεRI receptors are of human origin.
 15. The fusion molecule of claim 14wherein said first polypeptide sequence comprises a an amino acidsequence having at least 85% identity with a native human IgG heavychain constant region sequence.
 16. The fusion molecule of claim 15wherein said IgG is selected from the group consisting of IgG₁, IgG₂,IgG₃, and IgG₄.
 17. The fusion molecule of claim 15 wherein said nativehuman IgG heavy chain constant region sequence is the native human IgGheavy chain constant region sequence of SEQ ID NO:
 2. 18. The fusionmolecule of claim 17 wherein said first polypeptide sequence comprisesan amino acid sequence having at least 85% identity to the amino acidsequence of SEQ ID NO:
 3. 19. The fusion molecule of claim 18 whereinsaid first polypeptide sequence comprises an amino acid sequence havingat least 90% identity to the amino acid sequence of SEQ ID NO:
 3. 20.The fusion molecule of claim 19 wherein said first polypeptide sequencecomprises an amino acid sequence having at least 95% identity to theamino acid sequence of SEQ ID NO:
 3. 21. The fusion molecule of claim 20wherein said first polypeptide sequence comprises an amino acid sequencehaving at least 98% identity to the amino acid sequence of SEQ ID NO: 3.22. The fusion molecule of claim 21 wherein said first polypeptidesequence comprises at least part of the CH2 and CH3 domains of a nativehuman IgG, constant region.
 23. The fusion molecule of claim 22 whereinsaid first polypeptide sequence additionally comprises at least part ofthe hinge of a native human IgG₁ constant region.
 24. The fusionmolecule of claim 23 wherein said first polypeptide sequence comprisesat least part of the hinge, CH2 and CH3 domains of a native human IgG,heavy chain constant region, in the absence of a functional CHI region.25. The fusion molecule of claim 1 wherein said first polypeptidesequence comprises an amino acid sequence encoded by a nucleic acidhybridizing under stringent conditions to at least a portion of thecomplement of the IgG heavy chain constant region nucleotide sequence ofSEQ ID NO:
 1. 26. The fusion molecule of claim 3 wherein said first andsecond polypeptide sequences are functionally connected through alinker.
 27. The fusion molecule of claim 26 wherein said linker is apolypeptide linker.
 28. The method of claim 27 wherein said polypeptidelinker sequence consists of about 5 to about 25 amino acid residues. 29.The method of claim 1, wherein said fusion molecule comprises at leastone amino-terminal ubiquitination target motif.
 30. The method of claim1, wherein said fusion molecule comprises at least one proteasomeproteolysis signal, wherein said signal is selected from the groupconsisting of large hydrophobic amino acid residues, basic amino acidresidues and acidic amino acid residues.
 31. The method of claim 27,wherein said polypeptide linker comprises at least one proteasomeproteolysis signal, wherein said signal is selected from the groupconsisting of large hydrophobic amino acid residues, basic amino acidresidues and acidic amino acid residues.
 32. The method of claim 27wherein said polypeptide linker sequence comprises at least oneendopeptidase recognition motif.
 33. The method of claim 27 wherein saidpolypeptide linker sequence comprises a plurality of endopeptidaserecognition motifs.
 34. The method of claim 32 wherein saidendopeptidase recognition motif is selected from the group consisting ofcysteine, aspartate and asparagine amino acid residues.
 35. An isolatednucleic acid molecule encoding a fusion molecule of claim
 1. 36. Anisolated nucleic acid molecule encoding a fusion molecule of claim 3.37. A vector comprising and capable of expressing a polypeptide encodedby a nucleic acid molecule of claim
 35. 38. A host cell transformed withthe nucleic acid molecule of claim
 35. 39. A host cell transformed withthe nucleic acid molecule of claim
 36. 40. A pharmaceutical compositioncomprising a fusion molecule of claim 1 in admixture with apharmaceutically acceptable excipient.
 41. A pharmaceutical compositioncomprising a fusion molecule of claim 3 in admixture with apharmaceutically acceptable ingredient.
 42. An article of manufacturecomprising a container, a fusion molecule of claim 1 within thecontainer, and a label or package insert on or associated with thecontainer.
 43. An article of manufacture comprising a container, afusion molecule of claim 3 within the container, and a label or packageinsert on or associated with the container.
 44. The article ofmanufacture of claim 42 wherein said label or package insert comprisesinstructions for the treatment or prevention of an immune disease.
 45. Amethod for the treatment of an autoimmune disease in a subject,comprising administering an effective amount of at least one fusionmolecule of claim 3 to said subject diagnosed with or at risk ofdeveloping said autoimmune disease.
 46. The method of claim 45comprising multiple administration.
 47. The method of claim 45 whereinsaid subject is a human.
 48. The method of claim 45 wherein saidautoimmune disease is selected from the group consisting of rheumatoidarthritis, type-I diabetes mellitus, and multiple sclerosis.
 49. Themethod of claim 48 wherein said autoantigen is selected from the groupconsisting of rheumatoid arthritis autoantigen, multiple sclerosisautoantigen, autoimmune type I diabetes mellitus autoantigen, andportions thereof.
 50. The method of claim 49 wherein said autoantigen isselected from the group consisting of myelin basic protein (MBP),proteolipid protein, myelin oligodendrocyte glycoprotein, αβ-crystallin,myelin-associated glycoprotein, Po glycoprotein, PMP22, 2′,3′-cyclicnucleotide 3′-phosphohydrolase (CNPase), glutamic acid decarboxylase(GAD), insulin, 64 kD islet cell antigen (IA-2, also termed ICA512),phogrin (IA-2β), type II collagen, human cartilage gp39 (HCgp39), andgp130-RAPS.
 51. A method for the prevention of symptoms resulting from atype I hypersensitivity reaction in a subject receiving immunotherapy,comprising administering at least one fusion molecule to said subject,wherein said fusion molecule comprises a first polypeptide sequencecapable of specific binding to a native IgG inhibitory receptorcomprising an immune receptor tyrosine-based inhibitory motif (ITIM),functionally connected to a second polypeptide sequence capable ofbinding directly, or indirectly through a third polypeptide sequence, toa native IgE receptor (FcεR), wherein said first and second polypeptidesequences are other than antibody variable region sequences and whereinsaid fusion molecule is not capable of T cell interaction prior tointernalization, wherein said second polypeptide comprises a sequenceselected from the group consisting of: a) at least a portion of anautoantigen, b) an allergen, and c) at least a portion of an IgEimmunoglobulin heavy chain constant region capable of binding to anative IgE receptor (FcεR).
 52. The method of claim 51, wherein saidsymptoms resulting from a type I hypersensitivity reaction comprise ananaphylactic response.
 53. The method of claim 51 wherein said firstpolypeptide comprises at least a portion of an IgG immunoglobulin heavychain constant region.
 54. The method of claim 51, wherein said thirdpolypeptide is an IgE class antibody.
 55. The method of claim 51,wherein said subject receiving immunotherapy is being treated for adisease selected from type I hypersensitivity-mediated disease andautoimmune disease.
 56. The method of claim 51, wherein said fusionmolecule is administered to said subject prior to said subject receivingimmunotherapy.
 57. The method of claim 51, wherein said fusion moleculeis co-administered to said subject with said immunotherapy.
 58. Themethod of claim 51, wherein said fusion molecule is administered aftersaid subject receives immunotherapy.
 59. A method for the prevention ofa type I hypersensitivity disease in a subject receiving immunotherapy,comprising administering at least one fusion molecule to said subject,wherein said fusion molecule comprises a first polypeptide sequencecapable of specific binding to a native IgG inhibitory receptorcomprising an immune receptor tyrosine-based inhibitory motif (ITIM),functionally connected to a second polypeptide sequence capable ofbinding directly, or indirectly through a third polypeptide sequence, toa native IgE receptor (FcεR), wherein said first and second polypeptidesequences are other than antibody variable region sequences and whereinsaid fusion molecule is not capable of T cell interaction prior tointernalization, and wherein said second polypeptide comprises asequence selected from the group consisting of: a) at least a portion ofan autoantigen, b) an allergen, and c) at least a portion of an IgEimmunoglobulin heavy chain constant region capable of binding to anative IgE receptor (FcεR).